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. 2022 May 11;14(5):1024.
doi: 10.3390/v14051024.

Unique Aggregation of Retroviral Particles Pseudotyped with the Delta Variant SARS-CoV-2 Spike Protein

Jennifer D Petersen  1 , Jianming Lu  2 , Wendy Fitzgerald  3 , Fei Zhou  4 , Paul S Blank  1 , Doreen Matthies  4 , Joshua Zimmerberg  1
Affiliations

Unique Aggregation of Retroviral Particles Pseudotyped with the Delta Variant SARS-CoV-2 Spike Protein

Jennifer D Petersen et al. Viruses. .

Abstract

Individuals infected with the SARS-CoV-2 Delta variant, lineage B.1.617.2, exhibit faster initial infection with a higher viral load than prior variants, and pseudotyped viral particles bearing the SARS-CoV-2 Delta variant spike protein induce a faster initial infection rate of target cells compared to those bearing other SARS-CoV-2 variant spikes. Here, we show that pseudotyped viral particles bearing the Delta variant spike form unique aggregates, as evidenced by negative stain and cryogenic electron microscopy (EM), flow cytometry, and nanoparticle tracking analysis. Viral particles pseudotyped with other SARS-CoV-2 spike variants do not show aggregation by any of these criteria. The contribution to infection kinetics of the Delta spike's unique property to aggregate is discussed with respect to recent evidence for collective infection by other viruses. Irrespective of this intriguing possibility, spike-dependent aggregation is a new functional parameter of spike-expressing viral particles to evaluate in future spike protein variants.

Keywords: SARS-CoV-2; aggregation; coronavirus; pseudotyped viral particle; spike protein; variant.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Negative stain TEM detects unique aggregation of PVs bearing the Delta spike. (A–D) Low magnification negative stain TEM overviews of PVs bearing SARS-CoV-2 spike variants (A) D614G, (B) Alpha, (C) Bald, or (D) Delta. Black boxes surround individual PVs; blue box indicates an aggregate of Delta PVs. To the right are enlarged views of individual unlabeled PVs and PVs immunogold labeled for spike protein. (A’) Example of a doublet of D614G PVs. White arrowheads show areas on PVs where a fringe of spike proteins is clearly visible. Yellow arrowheads indicate 10 nm immunogold particles labeling the spike S1 subunit, which appear as black dots. Small black arrows indicate lipoprotein-like particles. (E) Frequency of singles, multiples, and aggregates of eight or more PVs per variant.
Figure 2
Figure 2
Negative stain TEM of Delta and Delta AY.4.2 aggregates observed 4 h after harvest from producer cells. (A) Delta and Delta AY.4.2 PV aggregates representing range of sizes at the 4 h timepoint. The number of PVs estimated per aggregate is indicated in the upper right corner of each image and the area occupied by the aggregate indicated in the lower right corner. Two Delta AY.4.2 PV aggregates are labeled, all others shown are Delta PVs (unlabeled). (B) Areas boxed in A are shown enlarged to the right of each image. Blue arrows highlight spike tip interactions occurring between PVs at the periphery of aggregates. (C) Graphs show ordered lists of the estimated number of PVs and areas of aggregates. (D) Truncated Weibull Distribution for Delta Variant. Negative stain data at 4 h was fit by the general model: f(x) = (wblcdf(x,a,b) − wblcdf(7,a,b))/(1 − wblcdf(7,a,b)) where wblcdf is the Weibull Distribution and x is the PVs per aggregate, 7 is the truncation value, and a and b are the Weibull scale and shape parameters. Coefficients (with 95% confidence bounds) are a = 11.47 (8.35, 14.60) and b = 0.68 (0.57, 0.79). Goodness of fit measure Sum of Squared Error (SSE) = 0.009 and Standard Error of Regression (RMSE) = 0.021, and Resid. are the residuals or differences between the model fit and the data.
Figure 3
Figure 3
Negative stain TEM of Delta and Delta AY.4.2 aggregates 24 h after harvest from producer cells. (A) Images of aggregates of Delta and Delta AY.4.2 PVs representing a range of sizes observed after storage overnight at 4 °C. The area occupied by each aggregate is indicated in the lower right corner. One donut-shaped Delta AY.4.2 aggregate is labeled, all other aggregates are Delta PVs (unlabeled). All images are scaled the same and scale bar is indicated. (B) Graph shows an ordered list of the areas of Delta aggregates.
Figure 4
Figure 4
Flow cytometry and NTA detection of Delta PV aggregates. Representative flow cytometry of variant PVs conducted 24 h (A) and 7 days (B) post-harvest. A population of particles over 200 nm in size are present in the Delta PVs and are minimally (<10%) represented in the other PV variant samples. (C) NTA of the same samples of PVs at 24 h post-harvest shows about half as many particles in the single PV range in the Delta sample compared to the other variants, and detection of a larger proportion of PVs in aggregates.
Figure 5
Figure 5
Cryo-electron microscopy of spike variant PVs. (A) Examples of singles (top panel), doubles, or aggregates (lower panel) of each variant PV. Spike variant is identified above. Black arrows indicate lipoprotein-like particles. The average PV envelope diameters for each variant are 118.6 (1.1), 118.5 (1.2), 119.6 (0.9), 120.1 (1.3), 115.8 (0.8), and 118.4 (0.6) nm for Bald, D614G, Alpha, Omicron BA.1, Delta, and Delta AY.4.2, respectively (mean (SEM), n = 9, 33, 40, 18, 65, and 98). (B) Low magnification overviews of Delta and Delta AY.4.2 PV aggregates prepared 4 or 24 h post-harvest. Dashed box shows the zoomed-in area. (C) The log-normal cumulative distributions for Delta and Delta AY.4.2 clearly show aggregation over time for both variants. At 4 and 24 h, Delta aggregate sizes (longest axis) range from 0.28–2.39 and 4.20–36.0 µm, with log-normal parameters mu (sigma) −0.18 (0.56) and 2.56 (0.44), corresponding to means of 0.97 and 14.25 µm, respectively. There is no evidence for significant differences in aggregate sizes between Delta and Delta AY.4.2 for both 4 and 24 h.
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