Minimal system for assembly of SARS-CoV-2 virus like particles

SARS-CoV-2 virus is the causative agent of COVID-19. Here we demonstrate that non-infectious SARS-CoV-2 virus like particles (VLPs) can be assembled by co-expressing the viral proteins S, M and E in mammalian cells. The assembled SARS-CoV-2 VLPs possess S protein spikes on particle exterior, making them ideal for vaccine development. The particles range in shape from spherical to elongated with a characteristic size of 129 ± 32 nm. We further show that SARS-CoV-2 VLPs dried in ambient conditions can retain their structural integrity upon repeated scans with Atomic Force Microscopy up to a peak force of 1 nN.


Scientific Reports
| (2020) 10:21877 | https://doi.org/10.1038/s41598-020-78656-w www.nature.com/scientificreports/ was performed on VLPs by applying 3.5 μL of VLPs to a glow-discharged formvar-carbon coated EM grid (Ted Pella, Redding, CA) followed by two de-ionized water washes and staining with 1% uranyl acetate. Imaging was performed in a JOEL JEM1400-Plus microscope with an accelerating voltage of 120 keV. Western Blot analysis. After triple washing the cells with PBS, 293 T cells were suspended in 100ul of RIPA buffer (sc-24948, Santa Cruz, Dallas, Texas). 10 μl of VLPs or cell extracts were then denatured by Laemmli sample buffer (BioRad, Hercules, CA) with 5% BME and boiled at 95C for 10 min. The proteins were separated by SDS-PAGE and then transferred to a PVDF membrane (Millipore, Burlington, Massachusetts). Membranes were stained with 1:1000 dilution of Anti-SARS-CoV SΔ10 antibody ([1A9], GenTex, Irvine, CA) along with 1:1000 dilution of Anti-Membrane Protein (2019-nCoV) Polyclonal Antibody (NCV-M-005, eEnzyme, Gaithersburg, MD) and then immunoprobed with appropriate infrared secondary antibody. Both antibodies were confirmed to be specific to intended antigens (Fig. S3) Membrane was scanned with the Odyssey infrared imaging system (LI-COR, Lincoln, NE) according to the manufacturer's manual instruction at 700 nm and 800 nm.
AFM sample prep and experiments. AFM experiments were performed using Dimension Icon AFM with an MLCT-BIO-DC probe (Bruker, Santa Barbara, CA, USA) in PeakForce QNM in air mode at room temperature. Glass coverslips were functionalized with anti-S antibody as follows. Glass was first cleaned and functionalized with biotin-PEG-silane as previously described 15 . The surfaces were then incubated with neutravidin (Thermo Scientific Pierce Protein Biology, Waltham, MA, USA) followed by a brief dd-H20 wash to get rid of excess neutravidin and incubation with biotinylated anti-S antibody (11-2001-B, Abeomics, San Diego, CA, USA). Surfaces thus prepared were generally devoid of debris but sometimes had step-edge character suggesting that antibody coating was less than a full monolayer (Fig. S4). The VLPs suspended in assay buffer were then incubated with the functionalized surface and finally washed away via brief buffer exchange with dd-H20 followed by drying under Nitrogen gas flow. All incubations lasted 30 min at room temperature. Assay buffer: PBS, pH 7.

Results
VLP purifications were first assessed biochemically. Figure 1 (see also Fig. S1) shows western blot analysis of cell extracts as well as purified VLPs. The isolated VLPs have both M and S proteins as identified in western blots. S protein appears more highly enriched in cells vs VLPs suggesting that not all expressed S protein is released on VLPs. The isolated VLPs were further analyzed by running a 10-40% sucrose gradient and sequential The gel was performed simultaneously, scanned at 700 nm and 800 nm, then superimposed using ImageJ (NIH) color merge for greater clarity, followed by cropping and minimal contrast enhancement. S protein (red) and M protein (green) are seen in two middle lanes. S protein tends to aggregate at very high concentrations (Fig. S2) resulting in smeared signal above 250 kD. S protein cleavage product is seen at 75 kD however the band is better visible when imaging channels are shown separately (Fig. S6). Protein size standards are described in "Methods" and are seen in leftmost and rightmost lanes. All size standard bands are also annotated on the left. www.nature.com/scientificreports/ fractionation. VLPs were found to be within the density of 20-25% sucrose (Fig. S2). S protein bands show a cleavage product which runs at a lower molecular weight from full S protein as shown in Fig. 1 and more clearly visible in Fig. S2. In addition both S protein as well as M protein form complexes which survive during western blot analysis and run at considerably higher molecular weight; successive dilutions results in breakup of these higher molecular weight fractions to respective monomeric bands for each protein (Fig. S3). We further characterized the purified VLPs via electron microscopy. Figure 2 shows a representative image of the electron micrographs with the immobilized VLPs on the EM grids. VLPs can be identified via specific nanogold immunolabeling. Notably, nanogold labels not only decorate the VLPs but also proximal areas, suggestive of S protein dissociating from VLPs during surface deposition. The SARS-CoV-2 VLPs we have characterized have a size distribution of 129 ± 32 nm, consistent with the general characterization of prior coronaviruses with size distributions of 100-200 nm 4 . This is also consistent with prior reports for SARS-CoV VLPs, which were characterized with cryotomography and have size distribution of 79-224 nm (shape reportedly varied from nearly spherical to ellipsoidal with a 2 to 1 aspect ratio 9 ). Our measured VLPs are also consistent with limited electron microscopy data available for the fully infectious SARS-CoV-2 virions observed in patient samples 14 .
The structural integrity of VLPs can inform their practical applications as well as serve as an estimate of the stability of fully infectious SARS-CoV-2 virions. We therefore further characterized the VLPs on functionalized surfaces with atomic force microscopy (AFM) (Fig. 3 and Figs. S5 & S6). VLPs were independently imaged on multiple occasions, originating from multiple purification batches. The observed surface density was as high as ~ 100 per 10 μm square field of view for high-yield VLP batches. VLPs appeared as approximately spherically symmetric particles whose lateral diameter was in excess of 200 nm while heights of the particles varied between 50 and 60 nm. These shapes are consistent with VLP dimensions broadened laterally by imaging forces and tip curvature and reduced somewhat in height due to surface adhesion and possibly imaging forces. Although detailed characterization is subject of future work, we found that repeated imaging of VLPs with peak force of 1 nN led to gradual particle deformations: reduced particle heights and non-circular lateral cross-sections -consistent with VLP bursts (Fig. 3C and Fig. S6). This force is within the range of previously reported values (0.5-5 nN) for bursting various virus capsids 15,16 although most prior work has been done in liquid. Our observations represent an upper estimate of the rupture force for SARS-CoV-2. This demonstrates that SARS-CoV-2 VLPs can be disrupted by direct application of moderate mechanical perturbations and open the door for future studies of VLP mechanics and integrity.

Discussion
Expression of just M, S, and E proteins (Fig. 1) was sufficient for release of VLPs which were structurally competent for both harvesting and subsequent investigations (Figs. 2, 3). These results open the door for many further studies. They can serve as immunogenic agents in place of the full infectious virus. The VLPs can also serve to study virus interactions with proteins of interest (e.g. receptors)-to date most such studies were only possible at the single protein level or in the context of the infectious virus. In addition, artificial VLPs can now be used to study the mechanical properties of the SARS-CoV-2 virus as well as their dependence on environmental conditions. Finally, VLPs can also be used to develop and validate novel testing strategies for SARS-CoV-2. The expression methodology is robust and should require no modifications to create VLPs for most S, M, and E mutations found in native conditions 17 .