Christmas came early for me this year. My exciting gift: A new tool in the scientific arsenal to study SARS-CoV-2 variants using Virus-Like-Particles (VLPS).
This novel technology and tool developed in the laboratory of Dr. Vineet D. Menachery allows scientists to study variant characteristics quickly and without studying actual viruses in a biosafety level 3 (BSL3) laboratory which require scientists to have specific facilities and training to conduct the studies as safely as possible. More about Dr. Vineet D. Menachery can be viewed here.
A description of the technology was published in the journal Science on December 23, 2021 and is available below.
BRYAN A. JOHNSON AND VINEET D. MENACHERY
SCIENCE • 23 Dec 2021 • Vol 374, Issue 6575 • pp. 1557-1558 • DOI: 10.1126/science.abn3781
Although efforts have been made to understand the biology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a major focus has been on investigating genetic variation in the virus. However, progress is hampered by the need to perform experiments involving SARS-CoV-2 in biosafety level 3 (BSL3) laboratories, which require substantial training for safe operation. On page 1626 of this issue, Syed et al. (1) offer an alternative to using live virus, introducing a new SARS-CoV-2 virus-like particle (VLP) system. The authors innovate on previous VLP systems by incorporating a reporter construct to study infection (1, 2). Illustrating the system’s utility, they use VLPs to characterize mutations in SARS-CoV-2 variants of concern.
SARS-CoV-2 VLPs are created by expressing the four structural proteins—spike, membrane, envelope, and nucleocapsid—in a packaging cell line (2). Upon expression, VLPs consisting of these four proteins and a lipid membrane self-assemble and are released from the cell (3). Despite resembling SARS-CoV-2 morphologically, traditional VLPs cannot be used to study the effect of a mutation on fitness because they lack genetic material to deliver to target cells (2). Syed et al. introduced a key innovation. They first identified the SARS-CoV-2 packaging signal, a genetic marker used to identify full-length genomes for packaging into the virion (4). This packaging signal was incorporated into the 3’ untranslated region of a luciferase reporter plasmid, causing the resulting transcripts to be packaged within VLPs. Syed et al. show that VLPs deliver these luciferase reporters to target cells, allowing the resulting signal to be used as a proxy for SARS-CoV-2 infection. Thus, the effects of particular mutations on the strength of the luciferase signal can be used to determine modulation of SARS-CoV-2 infection (see the figure).
In the broader context of studying SARS-CoV-2 genetic variation, VLPs represent a middle ground between two commonly used methodologies: infectious clones and pseudovirus vectors. SARS-CoV-2 infectious clones are the gold standard because they create recombinant virus, incorporating mutations anywhere in the genome (5). However, using SARS-CoV-2 infectious clones is technically challenging and creates live SARS-CoV-2 that requires BSL3 laboratories for study. This limits the use of SARS-CoV-2 infectious clones to laboratories with access to such facilities and willingness to invest in developing a specialized skill set.
Pseudovirus systems are the leading alternative to using SARS-CoV-2 infectious clones. In these systems, SARS-CoV-2 spike protein is expressed in cells along with a noncoronavirus packaging system and a reporter gene, with the most common being lentivirus-based (6). Like the VLPs developed by Syed et al., pseudoviruses self-assemble, incorporating spike proteins on their surface and packaging reporter messenger RNA (mRNA) (6). The primary advantage of pseudovirus systems is their ease of use, allowing rapid analysis of spike mutations. Pseudoviruses can be generated in the widely available 293T cell line by simply expressing a small number of proteins (6). Additionally, because pseudoviruses replace replication genes, they do not undergo continued amplification in target cell lines (6). This makes them safe to use in BSL2 laboratories, which are available to most researchers. However, the only SARS-CoV-2 protein incorporated into pseudoviruses is spike. Because substantial genetic variation occurs outside of spike, the pseduovirus systems have limited applicability to study SARS-CoV-2 variants.
The SARS-CoV-2 VLPs used by Syed et al. offer researchers several advantages over pseudoviruses. Rather than relying on the packaging machinery of another virus, VLPs use SARS-CoV-2 proteins and recapitulate packaging, assembly, and release, as occurs in genuine virus infection (3). In principle, this allows the effects of variant mutations on these processes to be studied. Similarly, because all four structural proteins are incorporated into SARS-CoV-2 VLPs, additional genetic variation can be captured. Like pseudoviruses, VLPs do not undergo subsequent rounds of replication, allowing them to be used safely in BSL2 laboratories.
Illustrating the utility of SARS-CoV-2 VLPs, Syed et al. characterized several nucleocapsid mutations. SARS-CoV-2 nucleocapsid is a hotspot for coding mutations, particularly within its serine-rich (SR) motif (7, 8). Although its exact function is unclear, the SR motif has many phosphorylated amino acids and is located within a region of intrinsic structural disorder (7, 9, 10). Using their SARS-CoV-2 VLP system, Syed et al. analyzed the effects of several common nucleocapsid mutations and found that several enhanced infection, including those present in the Alpha, Gamma, and Delta variants (7). These data are consistent with findings using SARS-CoV-2 infectious clones (1, 11).
The finding that nucleocapsid mutations enhance SARS-CoV-2 infection has important implications. To date, most studies of SARS-CoV-2 genetic variation have focused on spike (12). This is understandable, because spike binds to the host cell receptor angiotensin-converting enzyme 2 (ACE2), and is thus the primary determinant of infection (13). Additionally, because spike is the target of available vaccines, determining if mutations affect protection is a pressing question (12). However, recent studies suggest that nucleocapsid mutations lead to enhanced virulence and fitness, highlighting the need to characterize genetic variation elsewhere in the viral genome (11). Because SARS-CoV-2 VLPs recapitulate enhancement of infection by these nucleocapsid mutations, they can be used to characterize mutations in emerging variants, such as deletion of amino acids 31 to 33 in the nucleocapsid protein of the Omicron variant.
Although a promising platform, there are limitations of this SARS-CoV-2 VLP system. Only the four structural proteins are present. Thus, like pseudoviruses, the scope of variation that can be captured is limited. For example, variant mutations in the viral replication machinery cannot be examined with VLPs (14). Additionally, while allowing for safe use in BSL2 laboratories, the inability of VLPs to undergo continued replication makes them unsuitable to study virulence or transmission. Furthermore, although data presented by Syed et al. suggest that enhancement of infection by VLPs and live SARS-CoV-2 are correlated, additional work is needed to determine how closely VLPs model infection. As SARS-CoV-2 evolves, it is critical that the effects of new mutations are characterized.
Acknowledgments
V.D.M. is funded by the National Institutes of Health and National Institute of Allergy and Infectious Diseases (grants AI153602, 1R21AI145400, and R24AI120942). V.D.M. has filed a patent on the reverse genetic system and reporter SARS-CoV-2.
References and Notes
1. A. M. Syed et al., Science 374, 1626 (2021).
2. H. Swann et al., Sci. Rep. 10, 21877 (2020).
3. P. S. Masters, Adv. Virus Res. 66, 193 (2006).
4. P.-K. Hsieh et al., J. Virol. 79, 13848 (2005).
GO TO REFERENCECROSSREFPUBMEDGOOGLE SCHOLAR
5. X. Xie et al., Cell Host Microbe 27, 841 (2020).
GO TO REFERENCECROSSREFPUBMEDGOOGLE SCHOLAR
6. M. Chen, X.-E. Zhang, Int. J. Biol. Sci. 17, 1574 (2021).
7
Q. Ye et al., Protein Sci. 29, 1890 (2020).
8. J. A. Plante et al., Cell Host Microbe 29, 508 (2021).
GO TO REFERENCECROSSREFPUBMEDGOOGLE SCHOLAR
9. M. Bouhaddou et al., Cell 182, 685 (2020).
GO TO REFERENCECROSSREFPUBMEDGOOGLE SCHOLAR
10. A. D. Davidson et al., Genome Med. 12, 68 (2020).
GO TO REFERENCECROSSREFPUBMEDGOOGLE SCHOLAR
11. B. A. Johnson et al., bioRxiv2021).
12. W. T. Harvey et al., Nat. Rev. Microbiol. 19, 409 (2021).
13. C. B. Jackson et al., Nat. Rev. Mol. Cell Biol. (2021).
14. J. L. Mullen et al., https://outbreak.info/ (2020).