Rapid assessment of SARS-CoV-2-evolved variants using virus-like particles

Abstract

Efforts to determine why new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants demonstrate improved fitness have been limited to analyzing mutations in the spike (S) protein with the use of S-pseudotyped particles. In this study, we show that SARS-CoV-2 virus-like particles (SC2-VLPs) can package and deliver exogenous transcripts, enabling analysis of mutations within all structural proteins and at multiple steps in the viral life cycle. In SC2-VLPs, four nucleocapsid (N) mutations found universally in more-transmissible variants independently increased messenger RNA delivery and expression ~10-fold, and in a reverse genetics model, the serine-202→arginine (S202R) and arginine-203→methionine (R203M) mutations each produced >50 times as much virus. SC2-VLPs provide a platform for rapid testing of viral variants outside of a biosafety level 3 setting and demonstrate N mutations and particle assembly to be mechanisms that could explain the increased spread of variants, including B.1.617.2 (Delta, which contains the R203M mutation).

Fig. 1.. Design and characterization of SC2-VLPs.

(A) Schematic of SARS-CoV-2 and SC2-VLP design and location of RNA packaging sequence T20. (B) Process for generating and detecting luciferase-encoding SC2-VLPs. Numbers below plasmid maps indicate ratios used for transfection. (C) Induced luciferase expression measured in receiver cells (293T-ACE2/TMPRSS2) from “standard” SC2-VLPs containing S, M, N, E, and luciferase-T20 transcript, as well as VLPs lacking each one of the components. (D) N- or C-terminal 2× Strep-tag on M abrogates vector-induced luciferase expression. (E) Optimal luciferase expression requires a narrow range of spike plasmid concentrations corresponding to $\raisebox{1ex}{$1$}\!\left/ \!\raisebox{-1ex}{$500$}\right.$ of the total plasmid mass used for transfection. (F) Schematic for purification of SC2-VLPs. (G) Western blot showing S and N in pellets purified from standard SC2-VLPs and conditions that did not induce luciferase expression in receiver cells. (H) Schematic for sucrose gradient for separating SC2-VLPs. (I) Induced luciferase expression from sucrose gradient fractions of SC2-VLPs. gRNA, genomic RNA; IRES, internal ribosome entry site; Luc, luciferase; RLU, relative light units; S2, cleavage product of S; NS, nonspecific band. Error bars indicate SD with N = 3 independent transfections in each case.

Fig. 2.. RNA packaging into SC2-VLPs by SARS-CoV-2 sequences.

(A) Arrayed screen for determining the location of the optimal sequence for RNA packaging in SC2-VLPs. Two-kilobase tiled segments of the genome were cloned into the 3′ UTR of the luciferase plasmid. (B) Induced luciferase expression in receiver cells by SC2-VLPs containing different tiled segments from the SARS-CoV-2 genome. (C) Heatmap visualization of the data from (B), showing the locations of tiled segments relative to the SARS-CoV-2 genome. Color intensity indicates luminescence of receiver cells for each tile normalized to expression for a luciferase plasmid containing no insert. (D) Smaller segments of the genome were used to locate the optimal RNA packaging sequence. (E) Heatmap visualization of the data from (D). (F) Flow cytometry analysis of GFP expression in 293T ACE2/TMPRSS2 cells incubated with SC2-VLPs encoding GFP-PS9, GFP (no packaging sequence), or no VLPs. nts, nucleotides. Error bars indicate SD with N = 3 independent transfections in each case.

Fig. 3.. Effect of mutations in the S and N proteins on SC2-VLP–induced luminescence.

(A) Schematic for cloning and testing mutations observed in SARS-CoV-2 variants using SC2-VLPs. (B) Initial screen of 15 S mutants compared with a reference ancestral S containing the D614G mutation (termed WT). Details of mutations are listed in table S2. (C) Neutralization curve for SC2-VLPs generated using ancestral S and neutralized with the anti-S antibody MM43 (SinoBiological, catalog no. 40591). (D) Neutralization IC₅₀ of S variants using SC2-VLPs and MM43. (E) Initial screen of 15 N mutants compared with the reference Wuhan Hu-1 N sequence (WT). Details of mutations are listed in table S3. (F) Map of SARS-CoV-2 N domains showing the locations of observed mutations. Mutations that were observed to enhance signal are shown in bold. (B and E) Error bars indicate SD with N = 3 independent transfections in each case. Significance was determined by one-way analysis of variance and multiple comparisons using the Holm-Šídák test. **P < 0.01; ****P < 0.0001. (D) Error bars indicate 95% confidence intervals derived from curve fitting in GraphPad Prism. NTD, N-terminal domain; CTD, C-terminal domain. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; R, Arg; S, Ser; T, Thr; W, Trp; and Y, Tyr.

Fig. 4.. Impact of mutations in SARS-CoV-2 N on RNA packaging and viral titer.

(A) Luciferase expression in receiver cells from six N mutants retested after preparation in a larger batch. (B) Relative N-expression of selected mutants in packaging cells normalized to WT, with glyceraldehyde-3-phosphate dehydrogenase as a loading control. (C) Western blot (protein) and Northern blot (RNA) of VLPs generated using N-mutants purified by ultracentrifugation. Lentivirus was added before ultracentrifugation to allow use of p24 as an internal control. (D) Schematic for the reverse genetics system used to generate mutant SARS-CoV-2. (E) RT-qPCR of supernatant collected from A549-ACE2 cells infected with WT and mutant SARS-CoV-2 at MOI of 0.1 at 24, 48, and 72 hours after infection. (F) Representative plaques and (G) quantification of infectious viral titers from the same experiment. Error bars indicate SD with N = 3 independent transfections or infections in each case. Significance was determined by one-way analysis of variance and multiple comparisons using Holm-Šídák test. **P < 0.01; ****P < 0.0001; NS, not significant. F1 to F7, fragments 1 to 7; IVT, in vitro transcription; PFU, plaque-forming units.