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We tested for SC2-VLP production by co-transfecting the transfer plasmid into packaging cells (293T) along with plasmids encoding the viral structural proteins ( Fig. We designed a transfer plasmid encoding a luciferase transcript containing T20 within its 3′ untranslated region (UTR). The identification of such a SARS-CoV-2 cis-acting RNA element is required to create SARS-CoV-2 VLPs (SC2-VLPs) that incorporate and deliver engineered transcripts by this mechanism.īased on the reported packaging sequences for related viruses, including Murine Hepatitis Virus and SARS-CoV-1, we hypothesized that the SARS-CoV-2 packaging signal might reside within a region we termed “T20” (nt 20080-22222) encoding non-structural protein 15 (nsp15) and nsp16 ( Fig.
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During viral assembly, the N protein is thought to recognize one or more RNA structures within ORF1ab, enabling the full viral genome that contains this sequence to be packaged to the exclusion of viral subgenomic and host transcripts ( 19). A key requirement for such VLPs to deliver reporter transcripts into cells is the recognition of a cis-acting RNA sequence that triggers packaging. These VLPs appear to have similar morphology to infectious viruses and have been proposed as vaccine candidates ( 18).
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Previous studies have shown that co-expression of only the structural proteins of coronaviruses generates VLPs containing all four structural proteins ( 12– 17).
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We reasoned that a process mimicking viral assembly to package and deliver reporter transcripts would simplify the analysis of successful virus production, budding and entry.