Single-virus assay reveals membrane determinants and mechanistic features of Sendai virus binding

Abstract

Sendai virus (SeV, formally murine respirovirus) is a membrane-enveloped, negative-sense RNA virus in the Paramyxoviridae family and is closely related to human parainfluenza viruses. SeV has long been utilized as a model paramyxovirus and has recently gained attention as a viral vector candidate for both laboratory and clinical applications. To infect host cells, SeV must first bind to sialic acid glycolipid or glycoprotein receptors on the host cell surface via its hemagglutinin-neuraminidase (HN) protein. Receptor binding induces a conformational change in HN, which allosterically triggers the viral fusion (F) protein to catalyze membrane fusion. While it is known that SeV binds to α2,3-linked sialic acid receptors, and there has been some study into the chemical requirements of those receptors, key mechanistic features of SeV binding remain unknown, in part because traditional approaches often convolve binding and fusion. Here, we develop and employ a fluorescence microscopy-based assay to observe SeV binding to supported lipid bilayers (SLBs) at the single-particle level, which easily disentangles binding from fusion. Using this assay, we investigate mechanistic questions of SeV binding. We identify chemical structural features of ganglioside receptors that influence viral binding and demonstrate that binding is cooperative with respect to receptor density. We measure the characteristic decay time of unbinding and provide evidence supporting a "rolling" mechanism of viral mobility following receptor binding. We also study the dependence of binding on target cholesterol concentration. Interestingly, we find that although SeV binding shows striking parallels in cooperative binding with a prior report of Influenza A virus, it does not demonstrate a similar sensitivity to cholesterol concentration and receptor nanocluster formation.

Figure 1

Overview of single-virus-binding assay design and validation data. (A) shows a schematic of the assay design. Supported lipid bilayers (SLBs) are self-assembled inside a microfluidic device. Sendai virions, membrane labeled with Texas red-DHPE (TR) or R18, are introduced into the flow cell, where they can bind to receptors in the SLB. Binding and, in limited cases, fusion are observed and quantified by fluorescence microscopy. (B) shows a linear relationship between the viral concentration added to the flow cell, and the relative number of virions bound. In these measurements, the SLB contained 2% GD1a receptor and the viral concentration ranged from 0.025 to 0.1 nM. Error bars are ±standard error of ≥3 sample replicates, with propagated relative error as described in the supporting material. (C) depicts the relative number of virions bound to SLBs containing either 2% GD1a or no receptor. Very little binding is observed in SLBs without receptor. Error bars are ±standard error of ≥3 sample replicates, with propagated relative error as described in the supporting material. (D) shows the fraction of spots bound to various SLBs, which show positive immunofluorescence (IF) labeling by anti-hemagglutinin-neuraminidase (HN; 1A6) antibody, followed by Alexa 488-labeled secondary antibody. Colocalization between Alexa 488 and the membrane label in the particles (TR or R18) was used to determine whether a particle was IF positive. “No SLB” indicates that particles were attached non-specifically to the glass coverslip instead of a SLB, followed by surface passivation by 30 g/L bovine serum albumin. “0.5× R18” indicates that viral particles were labeled with R18 at 0.5× concentration (see Materials and methods for labeling details); all other data in this panel were collected with TR-labeled particles. Error bars are ±standard error of ≥3 sample replicates, with ≥10 separate image locations in each sample; the “0.5× R18” sample had 1 sample replicate, and error shown is standard deviation of 10 separate image locations. (E) shows the sensitivity of Sendai virus binding to 2% GD1a SLBs following pre-treatment of the virus with 1A6 antibody. Virus labeled with R18 (1×) was incubated with 1A6 antibody at the concentrations shown for 30 min on ice prior to injection into the flow cell. The blue line shows the best fit to a sigmoid curve; IC₅₀ = 1.3 ± 0.1 μg/mL. Error bars are ±standard deviation of 6–10 separate image locations within each sample. To see this figure in color, go online.

Figure 2

Chemical structure of gangliosides in SLBs directly modulates Sendai virus (SeV) binding. Single-virus-binding measurements were performed to SLBs containing 2% GM3, GM1, GD1a, GQ1b, or no receptor. (A) shows the chemical structure of each receptor used. Sialic acids are highlighted in blue. Note that GM1, GD1a, and GQ1b each possesses the same ganglio-tetrose backbone, whereas GM3 lacks the final 2 sugars of the tetrose backbone. (B) shows the relative number of virions bound to SLBs with the different ganglioside receptors. Binding measurements are shown relative to 2% GD1a. Error bars are ±standard error of ≥4 sample replicates, with propagated error as described in the supporting material. To see this figure in color, go online.

Figure 3

SeV exhibits cooperative binding to SLBs with either GD1a or GQ1b. Single-virus-binding measurements were performed to SLBs with varying concentrations of either (A) GD1a or (B) GQ1b. Solid lines show fits of the data to a Hill model binding curve (Eq. 1) to quantify the extent of cooperativity. The relative number of virions bound in both panels is calculated relative to 2% GD1a, and for ease of comparison are shown overlaid in (C). Error bars are ±standard error of ≥3 sample replicates, with propagated relative error as described in the supporting material. Best-fit parameters are shown in Table 1. To see this figure in color, go online.

Figure 4

SeV binding to SLBs with GD1a is insensitive to cholesterol concentration. Single-virus-binding measurements were performed on SLBs with varying concentrations of cholesterol and either 1% GD1a (yellow bars) or 2% GD1a (red bars). The SLBs also contained 20% DOPE, 0.05% Oregon green-DHPE, and the remainder POPC (47.95%–78.95%). The relative number of virions bound was calculated relative to 2% GD1a, 10% cholesterol. Within a set concentration of receptor, little difference in binding was observed across the range of cholesterol concentrations tested. By comparison, influenza A virus in Ref. (23) shows an ∼2-fold linear increase in binding to similar SLBs across the same range of cholesterol. Error bars are ±standard error of ≥3 sample replicates, with propagated relative error as described in the supporting material. To see this figure in color, go online.

Figure 5

Antibody treatment following SeV binding increases the rate of viral unbinding. Shown are example unbinding curves for SeV bound to SLBs with 2% GD1a. Following virus binding, antibody solution (or a no-antibody control solution) was introduced into the flow cell, and viral unbinding from the SLB was observed over 1 h. Shown are example data of the fraction of viruses bound over time in the presence of (A) no antibody, (B) anti-HN (1A6) at 20 μg/mL, and (C) anti-HN (3G12) at 20 μg/mL. Red lines represent best fits to an exponential decay curve (Equation S4) to estimate the characteristic decay time (τ_unbind). To see this figure in color, go online.

Figure 6

Distribution of diffusion coefficients for mobile virions on supported lipid bilayers. Diffusion coefficients were calculated from single-particle tracking (SPT) traces of bound virions in time-lapse micrographs over 25 min. Inset shows sample SPT traces; 50% of particles were immobile (D < 1.2 × 10⁻⁵ μm²/s, the lower limit of detectable diffusion); these are not shown in the distribution. The vast majority (∼80%) of mobile virions exhibited slow diffusion D < 0.025 μm²/s. The SLB contained 2% GD1a. Total number of particles analyzed was 1,035. To see this figure in color, go online.