Novel Mode of nanoLuciferase Packaging in SARS-CoV-2 Virions and VLPs Provides Versatile Reporters for Virus Production
Abstract
:1. Introduction
2. Materials and Methods
2.1. Plasmid Production
2.2. Expression and Purification of SARS-CoV-2 VLPs
2.2.1. Cells
2.2.2. Transfection
2.2.3. VLP Collection
2.2.4. VLP Concentration and Purification with Sucrose Cushion Ultracentrifugation
2.2.5. Gradient Purification of VLPs
2.3. Western Blotting
2.4. RNA Extraction and qRT-PCR
2.5. Microscopy
2.6. nanoLuciferase VLP Measurement
2.7. Virus Growth, Purification, and nanoLuciferase Measurement
2.8. Protease Protection Assay
2.9. Spike Mini-Binder Pull-Down
2.10. siRNA Treatments
2.11. Inhibitor Treatments
3. Results
3.1. Development of SARS-CoV-2 Virus-Like Particles Bearing a Reporter mRNA
3.2. The nanoLuciferase Protein Is Packaged and Secreted in SARS-CoV-2 VLPs
3.3. Coronaviruses but Not Flaviviruses Package nanoLuciferase Reporters in Their Viral Particles
3.4. nLuc Is Packaged in SARS-CoV-2 Virus Particles
3.5. Size Constrains Non-Specific Protein Packaging into Coronavirus Particles
3.6. Subcellular Location Constrains nLuc Packaging into Coronaviruses
3.7. nanoLuciferase Activity Reports on SARS-CoV-2 Replication Levels
4. Discussion
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Primer Name | Sequence |
---|---|
EGFP-qRTPCR-FWD | TCGCCGACCACTACCAGCAGAA |
EGFP-qRTPCR-REV | CGCGCTTCTCGTTGGGGTCTTT |
NSP15-qRTPCR_FWD | TTTGGGTGTGGACATTGCTGCT |
NSP15-qRTPCR_REV | ACAGTGAGTGGTGCACAAATCGT |
GAPDH-qRTPCR_FWD | TTCGACAGTCAGCCGCATCTTCTT |
GAPDH-qRTPCR_REV | GCCCAATACGACCAAATCCGTTGA |
nLuc-12xMBL-FL_FWD | ATGGTCTTCACACTCGAAGATTTCG |
nLuc-12xMBL-FL_REV | CAGGTTCAGGGGGAGGTGTG |
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Gullberg, R.C.; Frydman, J. Novel Mode of nanoLuciferase Packaging in SARS-CoV-2 Virions and VLPs Provides Versatile Reporters for Virus Production. Viruses 2023, 15, 1335. https://doi.org/10.3390/v15061335
Gullberg RC, Frydman J. Novel Mode of nanoLuciferase Packaging in SARS-CoV-2 Virions and VLPs Provides Versatile Reporters for Virus Production. Viruses. 2023; 15(6):1335. https://doi.org/10.3390/v15061335
Chicago/Turabian StyleGullberg, Rebekah C., and Judith Frydman. 2023. "Novel Mode of nanoLuciferase Packaging in SARS-CoV-2 Virions and VLPs Provides Versatile Reporters for Virus Production" Viruses 15, no. 6: 1335. https://doi.org/10.3390/v15061335