UB Distinguished Professor University at Buffalo - SUNY, New York, United States
Introduction:: SARS-CoV-2 is the virus causing the pandemic COVID-19 [1]. Over the last 1-2 years, virus-like particles (VLPs) have been developed that mimic the authentic virus [2], and these have been applied in basic science BSL-1/-2 level studies [3]. While the original SARS-CoV-2 particles were made using a 4-plasmid mixture (‘4P’), we determined if these can be reduced to 3- (‘3P’) and 2- (‘2P’) plasmid systems. This could simplify wet-lab experimental workflows and increase particle infectivity. Additionally, we determined if the tropism of these VLPs could be tuned by varying the envelope protein. Finally, as the size of SARS-CoV-2 VLPs (100-120nm) is larger than lentivirus (90nm) and adeno-associated virus (25nm), we determined if large nucleic acid payloads may be carried using these synthetic vehicles.
Materials and Methods:: The SARS-CoV-2 virion contains 4 structural proteins: M (membrane), E (envelope), N (nucleocapsid) and S (Spike). Assembling these components in the “4P” VLP system, necessitates co-transfection of host cells with 4-plasmids: pcDNA3.1 N, pcDNA3.1 M-IRES-E, Spike plasmid, and plasmid encoding for the SARS-CoV-2 packaging signal (PS9) fused to reporter/gene of interest (GOI). The “3P” system is similar only N, M and E were combined into a single pcDNA3.1 N-T2A-M-IRES-E construct. In the two plasmid “2P” system a range of plasmids, promoter sequences and gene insert permutations were tested to assemble an optimal VLP construct that combined N, M, E and PS9-GOI in a single plasmid. Additionally, a range of GOI and envelope proteins were tested to study packaging capacity and VLP tropism. This was enabled using Western blot analysis of the component proteins, qRT-PCR to quantify PS9 incorporation and Cryo-TEM and NanoSight for sizing. Finally, a range of microscopy, flow cytometry, and luminescence based measurements were made to quantify viral entry/gene knockout efficiency.
Results, Conclusions, and Discussions:: Among the VLPs systems, “3P” displayed 4-5 fold greater infectivity compared to “4P”, due to superior co-expression of structural proteins in single cells (Fig. 1A). The tropism of these virions could be tuned by the incorporation of different envelope proteins (Fig. 2B). Due to this, virions bearing SARS-CoV-2 (2019) Spike or the original SARS-CoV (2003) Spike effectively targeted ACE2 expressing 293T and A549 cells. Virions expressing MERS-CoV (Middle East respiratory syndrome coronavirus) Spike protein entered DPP4/CD26 expressing cells. Additionally, virions carrying VSV-G (Vesicular stomatitis virus G glycoprotein) could infect all cell types due to the ability of VSV-G to bind ubiquitously expressed low-density lipoprotein receptors and phosphatidylserine. Viral infection with “2P’ virions was also possible with “2P.2” representing the best in this class (Fig. 1C). When the luciferase reporter was replaced with EGFP, we observed infection of ~80% of 293T cells bearing ACE2 by SARS-CoV-2 Spike VLPs (Fig. 1D). Besides, reporter genes, the VLPs were observed to efficiently package Cas9 protein and facilitate genome editing applications. Overall, this work developed a family of safe SARS-CoV-2 VLPs for the efficient, robust delivery of diverse payloads to heterologous cells. These new reagents will enable basic science mechanistic studies that analyze the effect of targeted mutations on viral function and tropism, and trans-complemented studies that study the molecular features regulating the infectivity of emerging Spike variants. As part of ongoing studies, we will also present the use of these novel particles for targeted delivery of nucleic acids in translational applications, and examples of non-integrative genome editing selectively in ACE2 expressing cells.
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References (Optional): : (1) Yang et al., eLife e61552, 2020. (2) Syed et al., Science374, 1626-1632, 2021. (3) Yang et al.,Science Advances, 8:eabq8678, 2021.
