Multi-virion infectious units arise from free viral particles in an enveloped virus

Abstract

Many animal viruses are enveloped in a lipid bilayer taken up from cellular membranes. Because viral surface proteins bind to these membranes to initiate infection, we hypothesized that free virions may also be capable of interacting with the envelopes of other virions extracellularly. Here, we demonstrate this hypothesis in the vesicular stomatitis virus (VSV), a prototypic negative-strand RNA virus composed of an internal ribonucleocapsid, a matrix protein and an external envelope¹. Using microscopy, dynamic light scattering, differential centrifugation and flow cytometry, we show that free viral particles can spontaneously aggregate into multi-virion infectious units. We also show that, following establishment of these contacts, different viral genetic variants are co-transmitted to the same target cell. Furthermore, virion-virion binding can determine key aspects of viral fitness such as antibody escape. In purified virions, this process is driven by protein-lipid interactions probably involving the VSV surface glycoprotein and phosphatidylserine. Whereas we found that multi-virion complexes occurred unfrequently in standard cell cultures, they were abundant in other fluids such as saliva, a natural VSV shedding route². Our findings contrast with the commonly accepted perception of virions as passive propagules and show the ability of enveloped viruses to establish collective infectious units, which could in turn facilitate the evolution of virus-virus interactions and of social-like traits³.

Fig. 1. Spontaneous aggregation of purified VSV virions.

Transmission electron micrographs of purified VSV virions (a-c), showing a virion suspension positively stained with uranyl acetate (a; arrows indicate contacts between virions), an ultrathin section of agar-encased virions (b; four virions are shown in trans section; the envelope bilayer is indicated with white dotted lines; the structure protruding from the envelope lipid bilayer, indicated with an arrow, probably corresponds to the envelope protein G), and a virion suspension negatively stained with phosphotungstic acid (c; the envelope protein is visible and appears to mediate virion-virion contacts, one of which is indicated). Additional micrographs are shown in Supplementary Fig. 1. Virions were subjected to DLS (d-g) by fixing virions immediately (untreated) or after incubation, as indicated. The z-average values obtained from six measurements are shown (d). Vertical grey lines indicate the mean z-average. Treatment effects were evaluated relative to the untreated group using t-tests (ns: P > 0.05; **: P < 0.01; ***: P < 0.001). e. Polydispersity indexes (pdi) obtained in the same set of measurements. Particle size distribution curves, expressed as the percentage total light scattering intensity (f) and raw auto-correlation data (g) are shown, where each curve is an average from the six measurements. h. Effect of aggregation on infectivity, as determined in BHK-21 cells by the plaque assay after incubating purified virions at 37°C for the indicated times. To assess PFU weight, virions were centrifuged directly (blue) or after 2 h incubation at 37°C (red), and the pellet and supernatant were titrated by the plaque assay (i; the pellet/supernatant titer ratio is represented). Pellet/supernatant titer ratios for increasing centrifugation forces (5 min spin) after incubating virions 1 h at 37°C are also shown (j). Error bars indicate the standard error of the mean from three assays.

Fig. 2. Functional implications of multi-virion infectious units.

Proof of concept for the co-transmission of genetic variants was obtained by flow cytometry using two fluorescently labelled viruses (a). Cells were inoculated with VSV-mCherry and VSV-GFP, incubated 7 h, and counted (10⁵ events per assay). Bottom: uninfected (blue), mCherry-positive (red), GFP-positive (green), and doubly fluorescent (purple) cell counts following direct inoculation with VSV-GFP and VSV-mCherry (untreated) or after co-incubation of the two virion types (0.5 h at 37°C, 2 h at 37°C, or 2 h at 25°C). Two independent assays (1, 2) are shown for each condition. Top: flow cytometry scatter plot from two of the experiments. The estimated fraction of total virions forming dual infectious units was inferred using a probabilistic model detailed in Supplementary Fig. 3. Data are provided in Supplementary Table 1. b. Effects of virion-virion binding on antibody escape. Plaque assays of the MAb-resistant (A3853C) and MAb-sensitive (WT) variants following pre-incubation of the two types of virions (37°C, 2 h) at different proportions (v:v): 5:0, 5:1, 5:5, 5:10, and 5:20. For each treatment, plaque assays were done in the absence of MAb (grey), adding MAb to the culture medium to inhibit plaque development (dark red) or adding MAb prior to cell inoculation to inhibit cell entry (light red). Data points indicate average titers (PFU/mL) obtained from three independent assays, and error bars indicate the standard error of the mean.

Fig. 3. Envelope protein-lipid interactions mediate virion-virion binding in VSV.

a. Co-fluorescence test. Cells were inoculated with VSV-mCherry and with pre-trypsinized VSV-GFP, and subsequently analyzed by flow cytometry (10⁵ events per assay). Counts of uninfected (blue), mCherry-positive (red), GFP-positive (green), and doubly fluorescent (purple) cells after direct inoculation with the viral mix (untreated) or after co-incubation of the two virion types (37°C, 2 h) are shown. Two independent assays (1, 2) are shown for each condition. b. DLS assays showing the effects of various treatments on virion aggregation. Purified virions were incubated at 37°C for 1 h alone (control) or in the presence of trypsin, PLC, PLA1A, annexin V, or mouse RAP. The change in z-average relative to the average change in z-average obtained for controls is shown, i.e. Δ(z-average treatment)/Δ(z-average control). All assays were performed in triplicate and the error bar represents the standard error of the mean.

Fig. 4. VSV virion-virion binding in cell culture media, plasma, and saliva.

a. In each of the fluids tested, ca. 10⁹ PFU/mL of each VSV-GFP and VSV-mCherry were co-incubated at 37°C for 2 h in FBS or human (h) plasma, 1 h in human saliva, or 15 min in cow (c) saliva, or not co-incubated (untreated). For one of the human salivas, the test was done both at 10⁹ PFU/mL (circle) and 10⁷ PFU/mL (cross). The amount of virion aggregation is represented as the fraction of virions forming dual PFUs estimated by the co-fluorescence test (see Methods and Supplementary Fig. 3; cell counts are available from Supplementary Table 1). For plasma and saliva, each data point corresponds to an individual donor (human/cow) and represents the average of two independent assays. For FBS, three independent assays are plotted. For untreated viruses, the estimated amount of virion aggregation fluctuated around zero (from –1.4% to 1.9% in six independent assays) and is shown as a single blue dot at the lower-limit of the plot range. b. Virion aggregation as a function of the final titer (i.e. after incubation), showing the correlation between titer loss and aggregation.