Journal: Journal of Virology

Article Title: Enhanced sialic acid engagement at physiological temperatures by reovirus σ1 mutants facilitates infection of breast cancer cells with low levels of high-affinity receptors

doi: 10.1128/jvi.00074-26

Figure Lengend Snippet: E0771 cells have reduced expression of the high-affinity reovirus attachment receptor JAM-A. E0771 and L929 cells were harvested using CellStripper, fixed with 4% paraformaldehyde (PFA), and analyzed by flow cytometry following immunostaining with monoclonal antibodies specific for SNA, JAM-A, or β1 integrin. Representative histograms (left) and corresponding quantification (right) show cell surface expression of ( A ) α2,6-linked sialic acids detected using fluorescently labeled SNA lectin in neuraminidase-treated (+) or untreated (−) cells, ( B ) JAM-A detected using murine JAM-A-specific antibodies (+) compared with isotype (Iso) controls, and ( C ) β1 integrin detected using β1 integrin-specific primary antibodies (+) compared with Iso controls. MFIs were normalized to the corresponding negative controls (neuraminidase-treated or isotype-stained cells) from a representative L929 experiment. Data represent mean ± SD ( n = 3). Statistical significance was determined by the two-way ANOVA with Tukey’s multiple comparisons test in GraphPad Prism v10.4 (ns = P > 0.05; * P < 0.05; ** P < 0.005; *** P < 0.001; and **** P < 0.0001).

Article Snippet: To quantify cell-surface receptor expression, cells were incubated with monoclonal antibodies specific for murine JAM-A (clone BV11, Millipore Sigma), murine β1 integrin (eBioscience), human JAM-A (CSTEM27, Thermo Fisher Scientific), or human β1 integrin (clone P5D2, DSHB).

Techniques: Expressing, Flow Cytometry, Immunostaining, Bioprocessing, Labeling, Staining

Journal: Journal of Virology

Article Title: Enhanced sialic acid engagement at physiological temperatures by reovirus σ1 mutants facilitates infection of breast cancer cells with low levels of high-affinity receptors

doi: 10.1128/jvi.00074-26

Figure Lengend Snippet: Mutations in the sialic acid-binding domain permit high-affinity, receptor-independent attachment by increasing binding to sialic acids at physiological temperature (37°C). ( A ) Representative western blot analysis of outer capsid proteins µ1C and σ3, detected using anti-reovirus polyclonal serum. ( B and C ) L929 ( B ) and E0771 ( C ) cells were treated with PBS or neuraminidase (+neuraminidase) for 1 h at 37°C to deplete cell surface sialic acids. Viruses indicated in the legend were incubated with cells for 1 h at 4°C or 37°C in the presence of NH 4 Cl, washed, and cells processed for flow cytometric analysis using σ3-specific antibodies. MFI reflects the level of cell-associated virus particles. ( D ) Virus-cell association was measured as in panel C , without neuraminidase treatment, using parental U937 cells or U937 cells deficient in sialic acids (U937-Sia - ). ( E ) Flow cytometric detection of α2,6-linked sialic acids on RBCs using fluorescently labeled SNA lectin. Representative histograms (left) show unstained RBCs (light gray), SNA-stained RBCs (red), and SNA-stained H1299 cells (dark gray). n = 3. ( F ) Flow cytometric detection of JAM-A on RBCs using a primary/secondary antibody system specific for hJAM-A. Representative histograms (left) show RBCs with secondary antibody only (light gray), primary/secondary-stained RBCs (red), and E0771+JAM cells (dark gray). n = 3. ( G ) Flow cytometric detection of β1 integrins on RBCs using primary/secondary antibodies specific for human β1 integrin. Representative histograms (left) show RBCs with secondary antibody only (light gray), primary/secondary-stained RBCs (red), and H1299 cells (dark gray). n = 3. ( H ) RBCs were incubated with particle-normalized T3D PL at serial dilutions starting at 1.9 × 10 5 particles for 1 h at 4°C or 37°C. Unbound virions were removed by PBS washes prior to fixation and immunostaining for outer capsid proteins, followed by flow cytometric analysis. n = 3. ( I–K ) RBCs were incubated with particle-normalized T3D PL or variant viruses at serial dilutions starting at 1.9 × 10 5 particles for 1 h at 4°C ( I and J ) or 37°C ( I and K ). Following removal of unbound virions by PBS washes, cells were fixed, immunostained for outer capsid proteins, and analyzed by flow cytometry. Absolute MFI values ( I ) were used to calculate the AUC for each virus across all independent experiments, normalized to T3D PL at the corresponding temperature for each independent experiment ( n = 3–5). ( L ) Levels of σ1 per virion for full-length T3D PLσ1-G196R were assessed by agarose gel electrophoresis (top) and quantitative serial dilution-based western blot analysis using anti-σ3 and anti-µ1 monoclonal antibodies and anti-σ1 tail polyclonal antibodies (middle). Bottom: relative average σ1 per virion calculated relative to T3D PL from five independent virus preparations based on σ1 to (σ3 + µ1) protein ratios determined by western blot analysis. Data represent mean ± SD. Statistical significance was determined using the one-way ANOVA with Tukey’s multiple comparisons test ( E, J, and K ) or the paired t -test ( F, G, and H ) in GraphPad Prism v10.4. (ns = P > 0.05; * P < 0.05; ** P < 0.005; *** P < 0.001; and **** P < 0.0001).

Article Snippet: To quantify cell-surface receptor expression, cells were incubated with monoclonal antibodies specific for murine JAM-A (clone BV11, Millipore Sigma), murine β1 integrin (eBioscience), human JAM-A (CSTEM27, Thermo Fisher Scientific), or human β1 integrin (clone P5D2, DSHB).

Techniques: Binding Assay, Western Blot, Incubation, Virus, Labeling, Staining, Immunostaining, Variant Assay, Flow Cytometry, Agarose Gel Electrophoresis, Serial Dilution, Bioprocessing

Journal: Journal of Virology

Article Title: Enhanced sialic acid engagement at physiological temperatures by reovirus σ1 mutants facilitates infection of breast cancer cells with low levels of high-affinity receptors

doi: 10.1128/jvi.00074-26

Figure Lengend Snippet: Mutational enhancement of σ1-mediated receptor binding and structural basis of sialic acid interaction. ( A ) Model depiction of relative binding strengths deduced from experimental mean AUCs between the sialic acid-binding domain (orange) to sialic acids (dark gray, Sia), the JAM-A-binding domain (circular head of σ1) to JAM-A (black), and the RGD domain (red) to β-integrin (light gray, βInt). Where “~” is indicated, relative binding strength was deduced by subtracting the total binding measured in the JAM-A-deficient condition from the domain-specific binding strength. ( B ) Structural models of the T3D PL σ1 body domain were generated using UCSF ChimeraX (v1.9). Wild-type (bordered) and mutant σ1 structures were created by introducing identified substitutions. Predicted hydrogen bonds with α2,3-linked sialic acid (PDB: 3S6X) were assessed using default cutoffs; a representative G196R rotamer shows novel hydrogen bonds between the arg196 and sialic acid (red arrow). ( C ) Amino acid sequence alignment of the σ1 body domain encompassing the sialic acid-binding pocket (NCBI). Mutations identified through passage (G196R, T193M, and N206H) are annotated alongside known sialic acid-binding residues (N198, R202, and P204).

Article Snippet: To quantify cell-surface receptor expression, cells were incubated with monoclonal antibodies specific for murine JAM-A (clone BV11, Millipore Sigma), murine β1 integrin (eBioscience), human JAM-A (CSTEM27, Thermo Fisher Scientific), or human β1 integrin (clone P5D2, DSHB).

Techniques: Binding Assay, Generated, Mutagenesis, Sequencing