simian virus 40  (ATCC)

Journal: Molecular Therapy Oncology

Article Title: Optimization of the VSV-G backbone for amino terminal fusion with nanobodies allowing its specific retargeting to HER2 receptors

doi: 10.1016/j.omton.2025.201065

Figure Lengend Snippet: Optimization of the VSV-G backbone to tolerate an amino-terminal fusion with the C11 nanobody (A) Schematic representation of G WT and the G C11 chimera. QF: inserted dipeptide; GGGGS x 2: flexible linker; TM: transmembrane domain; IV: intraviral domain. (B) Schematic of the experimental design to evaluate transport of VSV-G and its variants to the cell surface. (C) Left: Transport of G WT , G C11 , and G C11-opt to the cell surface of transfected HEK293T cells. G was detected on the surface of non-permeabilized cells using 8G5F11 mAb specific for VSV-G ectodomain and a secondary antibody conjugated to Alexa Fluor 488. Cells were analyzed by flow cytometry. Right: Median surface expression levels, measured using flow cytometry, of G C11 and G C11-opt normalized to median concentration of G WT on the surface of HEK293T cells. (D) Schematic of the experimental design to generate VSVΔG-GFP pseudotyped by G chimeras (top) and to measure their infectivity (bottom). (E) Incorporation of G WT , G C11 , and G C11-opt into VSVΔG-GFP viral particles assessed by western blot analysis using an anti-VSV-G and an anti-VSV M antibody. In the bar diagram, the G/M ratio in VSVΔG-GFP pseudotyped by G C11 and G C11-opt was normalized to that in VSVΔG-GFP pseudotyped by G WT . (F) Infectivity of VSVΔG-GFP pseudotyped with G WT , G C11 , and G C11-opt in HEK293T cells. VSVΔG-GFP viruses pseudotyped with WT or chimeric glycoproteins produced in the same conditions (see ) were used to infect HEK293T cells during 16 h. The percentage of infected cells was measured by counting GFP-expressing cells by flow cytometry and used to calculate the viral titer. (G) Experimental evolution of rVSV-G C11 recombinant virus in BSR cells. rVSV-G C11 was serially passaged in BSR cells, and infected cells were labeled using an anti-N antibody and a goat anti-mouse IgG secondary antibody conjugated to Alexa Fluor 488. DAPI was used to stain nuclei. The S422I (after P4) and H22N (after P7) mutations invaded the viral population. Arrowheads indicate foci of infected cells. (H) Growth curves of VSV WT and rVSV-G C11-opt . At each time point, the viral titer was measured using plaque assay. (I) Negative staining electron microcopy of rVSV-G C11-opt virions incubated at pH 7.5 and 5.5. Insets show the magnification of the viral membrane revealing the typical shape of VSV-G at high and low pH. (J) Binding of mCherry protein to rVSV-G C11-opt . Indicated viruses were incubated with mCherry at indicated pHs, centrifuged, and the pellet was analyzed by SDS-PAGE. L, N, and M indicate the positions of viral proteins; G WT and G C11-opt , the G proteins; and mCh, the precipitated mCherry. (K) Infectivity of VSVΔG-GFP pseudotyped with G WT , G C11 , G C11-opt , G C11-H22N , and G C11-S422I in HEK293T cells. VSVΔG-GFP viruses pseudotyped with WT or chimeric glycoproteins, produced in the same conditions, were used to infect HEK293T cells during 16 h. The percentage of infected cells was measured by counting GFP-expressing cells by flow cytometry and used to calculate the viral titer. In (C), (E), (F), (H), and (K), data points represent replicates of at least three independent experiments. Errors bars indicate SD. In (C) and (E), statistically significant differences between G WT , G C11 , and G C11-opt are indicated by asterisks (∗ p < 0.05; ∗∗ p < 0.005; ∗∗∗ p < 0.0005; ∗∗∗∗ p < 0.00005).

Article Snippet: BSR, a clone of BHK-21 (baby hamster kidney; ATCC CCL-10), and HEK293T (human embryonic kidney expressing simian virus 40 T antigen [SV40T]; ATCC CRL-3216) cells were grown in DMEM supplemented with 10% fetal calf serum (FCS).

Techniques: Transfection, Flow Cytometry, Expressing, Concentration Assay, Infection, Western Blot, Produced, Recombinant, Virus, Labeling, Staining, Plaque Assay, Negative Staining, Incubation, Membrane, Binding Assay, SDS Page

Journal: Molecular Therapy Oncology

Article Title: Optimization of the VSV-G backbone for amino terminal fusion with nanobodies allowing its specific retargeting to HER2 receptors

doi: 10.1016/j.omton.2025.201065

Figure Lengend Snippet: Hydrophobic residues at position 422 optimize VSV-G for nanobody insertion (A) Structural environment of residue S422 in the crystal structure of the pre-fusion conformation of VSV-G. (B) Mutation S422I stabilizes G C-terminal domain (CTD) through hydrophobic stacking with residues F424 and L430. (C) Infectivity of VSVΔG-GFP pseudotyped with G WT , G C11 , G C11-opt , G C11-opt-S422F , G C11-opt-S422L , G C11-opt-S422M , G C11-opt-S422V , and G C11-opt-S422G in HEK293T cells. VSVΔG-GFP viruses pseudotyped with G WT or chimeras, produced in the same conditions, were used to infect HEK293T during 16 h. The percentage of infected cells was measured by counting GFP-expressing cells by flow cytometry and used to calculate the viral titer. Data points represent replicates of independent experiments. Error bars indicate SD. Statistically significant differences with G C11 (respectively G WT ) are indicated by blue (respectively green) asterisks (∗ p < 0.05; ∗∗ p < 0.005; ∗∗∗ p < 0.0005; ∗∗∗∗ p < 0.00005; ns, not significant).

Article Snippet: BSR, a clone of BHK-21 (baby hamster kidney; ATCC CCL-10), and HEK293T (human embryonic kidney expressing simian virus 40 T antigen [SV40T]; ATCC CRL-3216) cells were grown in DMEM supplemented with 10% fetal calf serum (FCS).

Techniques: Residue, Mutagenesis, Infection, Produced, Expressing, Flow Cytometry

Journal: Molecular Therapy Oncology

Article Title: Optimization of the VSV-G backbone for amino terminal fusion with nanobodies allowing its specific retargeting to HER2 receptors

doi: 10.1016/j.omton.2025.201065

Figure Lengend Snippet: Retargeting of VSVΔG-GFP toward HER2-expressing cells (A) Optimization factor of the titer of VSVΔG-GFP pseudotyped with the chimeric G in the optimized backbone compared with VSVΔG-GFP pseudotyped with a chimera in a non-optimized backbone. VSVΔG-GFP viruses, produced in the same conditions, pseudotyped with chimeras in an optimized or non-optimized backbone were used to infect HEK293T cells during 16 h. The percentage of infected cells was measured by counting GFP-expressing cells by flow cytometry and used to calculate the viral titer. The histogram shows the mean difference between the log 10 of the titer of VSVΔG-GFP pseudotyped with the optimized chimera and the log 10 of the titer of VSVΔG-GFP pseudotyped with the non-optimized chimera. The factor of optimization is indicated in red for each chimera. (B) Expression of HER2 in HEK293T and HEK293T-HER2 KO cells. Cell lysates were analyzed by western blot using anti-HER2 and anti-tubulin antibodies. (C) Cell surface expression of HER2 in HEK293T and HEK293T-HER2 KO cells. Expression of HER2 was analyzed using flow cytometry with an anti-HER2 as a primary antibody and a secondary antibody conjugated to Alexa Fluor 488. The percentage of HER2-positive cells is indicated. (D) Flow cytometry graph showing the infectivity in HEK293T and HEK293T-HER2 KO cells of VSVΔG-GFP pseudotyped with G WT , G K47Q , G R354Q , or with optimized G chimeras fused to nanobodies H1–H4 (recognizing HER2) in which mutations K47Q or R354Q have been introduced or not. VSVΔG-GFP viruses pseudotyped with these glycoproteins were used to infect indicated cell lines during 16 h. The percentage of infected cells was measured by counting GFP-expressing cells by flow cytometry and is indicated on the top right of each graph. (E) Histogram showing the log 10 of the titer of viruses pseudotyped with mutated or chimeric Gs normalized to that pseudotyped with G WT in the indicated cell line. Each point corresponds to a single experiment as in (D). A value of 0 means that the titer of the virus pseudotyped with mutated or chimeric Gs is equal to the titer of the virus pseudotyped with G WT (in the indicated cell line). Viral titers were calculated from the percentage of cells expressing GFP. The log 10 of the titer of VSVΔG-GFP virus pseudotyped with G WT was 7.37 ± 0.34 in HEK293T and 7.39 ± 0.38 in HEK293T-HER2 KO cells ( n = 13). In (A) and (E), error bars represent the SD for experiments carried out at least in triplicate (see also for A). Statistically significant differences are indicated by asterisks (∗ p < 0.05; ∗∗ p < 0.005; ns, not significant).

Article Snippet: BSR, a clone of BHK-21 (baby hamster kidney; ATCC CCL-10), and HEK293T (human embryonic kidney expressing simian virus 40 T antigen [SV40T]; ATCC CRL-3216) cells were grown in DMEM supplemented with 10% fetal calf serum (FCS).

Techniques: Expressing, Produced, Infection, Flow Cytometry, Western Blot, Virus

Journal: Molecular Therapy Oncology

Article Title: Optimization of the VSV-G backbone for amino terminal fusion with nanobodies allowing its specific retargeting to HER2 receptors

doi: 10.1016/j.omton.2025.201065

Figure Lengend Snippet: Infectivity of lentiviruses pseudotyped with G K47Q , G R354Q , or by optimized G chimeras fused to nanobodies H1–H4 in which mutations K47Q or R354Q have been introduced or not (A) Schematic of the experimental design to generate lentivirus pseudotyped by G chimeras (G ∗ ) and to measure their infectivity. (B) Flow cytometry graph showing the transduction efficiency in HEK293T and HEK293T-HER2 KO cells of lentiviruses pseudotyped with G WT , G K47Q , G R354Q , or with optimized G chimeras fused to nanobodies H1–H4 (recognizing HER2) in which mutations K47Q or R354Q have been introduced or not. The percentage of transduced cells was measured by counting GFP-expressing cells by flow cytometry and indicated on the top right of each graph. (C) Histogram showing the log 10 of the titer of lentiviruses pseudotyped with the indicated G construct normalized with the one pseudotyped with G WT . Each point corresponds to a single experiment as in (B). A value of 0 means that the titer of the lentivirus pseudotyped with mutated or chimeric Gs is equal to the titer of the virus pseudotyped with G WT (in the same cell line). Lentiviral titers were derived from the percentage of cells expressing GFP. The log 10 of the titer of lentiviruses pseudotyped with G WT was 6.89 ± 0.22 in HEK293T and 6.83 ± 0.22 ( n = 13) in HEK293T-HER2 KO cells. (D) Comparison of the expression of HER2 in HEK293T and SKBR3 cells. Cell lysates were analyzed by western blot with anti-HER2 and anti-tubulin antibodies. The difference of migration of HER2 observed in SKBR3 is likely due to alternative splicing, which is common in cancer cell lines. , (E) Flow cytometry graph showing the transduction efficiency in SKBR3 cells of lentiviruses pseudotyped with G WT , G K47Q , G R354Q , or with optimized G chimeras fused to nanobodies H1–H4 (recognizing HER2) in which mutations K47Q or R354Q have been introduced. The percentage of transduced cells was measured by counting GFP-expressing cells by flow cytometry and indicated on the top right of each graph. (F) Histogram showing the log 10 of the titer of lentiviruses pseudotyped with the indicated G construct normalized with the one pseudotyped with G WT . Each point corresponds to a single experiment as in (E). A value of 0 means that the titer of the lentivirus pseudotyped with mutated or chimeric Gs is equal to the titer of the virus pseudotyped with G WT . Lentiviral titers were derived from the percentage of cells expressing GFP 48 h after transduction. The log 10 of the titer of lentiviruses pseudotyped with G WT was 5.8 ± 0.15 ( n = 10) in SKBR3 cells. In (C) and (F), error bars represent the SD for experiments carried out at least in triplicate. Statistically significant differences are indicated by asterisks (∗ p < 0.05; ∗∗ p < 0.005; ∗∗∗ p < 0.0005; ∗∗∗∗ p < 0.00005; ns, not significant). In (F), blue (resp. orange) asterisks indicate the significance of the difference between G K47Q (resp. G R354Q ) and the chimera having the same mutation.

Article Snippet: BSR, a clone of BHK-21 (baby hamster kidney; ATCC CCL-10), and HEK293T (human embryonic kidney expressing simian virus 40 T antigen [SV40T]; ATCC CRL-3216) cells were grown in DMEM supplemented with 10% fetal calf serum (FCS).

Techniques: Infection, Flow Cytometry, Transduction, Expressing, Construct, Virus, Derivative Assay, Comparison, Western Blot, Migration, Alternative Splicing, Mutagenesis

Journal: Molecular Therapy Oncology

Article Title: Optimization of the VSV-G backbone for amino terminal fusion with nanobodies allowing its specific retargeting to HER2 receptors

doi: 10.1016/j.omton.2025.201065

Figure Lengend Snippet: VSV pseudotyped with optimized G K47Q or G R354Q chimeras fused to nanobodies H1–H4 preferentially infect HEK293T WT cells (A) Schematic of the experimental design: A 1:1 mix of HEK293T and HEK293T-HER2 KO (the latter expressing the RFP protein) was infected with VSVΔG-GFP pseudotyped with the indicated G construct. (B) Representative biparametric flow cytometry graph showing the infectivity of VSVΔG-GFP pseudotyped with the indicated glycoprotein in each cell population ( x axis: intensity of GFP fluorescence i.e. infection; y axis: intensity of RFP i.e. cell line identity). (C) Selectivity index of VSVΔG-GFP pseudotyped with the chimeras. The selectivity index was calculated by dividing the proportion of infected HEK293T cells by the proportion of infected HEKT293-HER2 KO cells in the mix. Each data point corresponds to a single experiment. For each G construct, the mean of experiments carried out at least in triplicate is indicated. The error bars represent the SD.

Article Snippet: BSR, a clone of BHK-21 (baby hamster kidney; ATCC CCL-10), and HEK293T (human embryonic kidney expressing simian virus 40 T antigen [SV40T]; ATCC CRL-3216) cells were grown in DMEM supplemented with 10% fetal calf serum (FCS).

Techniques: Expressing, Infection, Construct, Flow Cytometry, Fluorescence