RESUMO
Replicons, derived from RNA viruses, are genetic constructs retaining essential viral enzyme genes while lacking key structural protein genes. Upon introduction into cells, the genes carried by the replicon RNA are expressed, and the RNA self-replicates, yet viral particle production does not take place. Typically, RNA replicons are transcribed in vitro and are then electroporated in cells. However, it would be advantageous for the replicon to be generated in cells following DNA transfection instead of RNA. In this study, a bacterial artificial chromosome (BAC) DNA encoding a SARS-CoV-2 replicon under control of a T7 promoter was transfected into HEK293T cells engineered to functionally express the T7 RNA polymerase (T7 RNAP). Upon transfection of the BAC DNA, we observed low, but reproducible expression of reporter proteins GFP and luciferase carried by this replicon. Expression of the reporter proteins required linearization of the BAC DNA prior to transfection. Moreover, expression occurred independently of T7 RNAP. Gene expression was also insensitive to remdesivir treatment, suggesting that it did not involve self-replication of replicon RNA. Similar results were obtained in highly SARS-CoV-2 infection-permissive Calu-3 cells. Strikingly, prior expression of the SARS-CoV-2 N protein boosted expression from transfected SARS-CoV-2 RNA replicon but not from the replicon BAC DNA. In conclusion, transfection of a large DNA encoding a coronaviral replicon led to reproducible replicon gene expression through an unidentified mechanism. These findings highlight a novel pathway toward replicon gene expression from transfected replicon cDNA, offering valuable insights for the development of methods for DNA-based RNA replicon applications.
Assuntos
Genes Reporter , Replicação do RNA , RNA Viral , Replicon , SARS-CoV-2 , Humanos , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Alanina/análogos & derivados , Cromossomos Artificiais Bacterianos/genética , COVID-19/virologia , COVID-19/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Regiões Promotoras Genéticas , Replicon/genética , Replicação do RNA/genética , RNA Viral/genética , RNA Viral/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Transfecção , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19. Though many COVID-19 vaccines have been developed, most of them are delivered via intramuscular injection and thus confer relatively weak mucosal immunity against the natural infection. Virus-Like Particles (VLPs) are self-assembled nanostructures composed of key viral structural proteins, that mimic the wild-type virus structure but are non-infectious and non-replicating due to the lack of viral genetic material. In this study, we efficiently generated SARS-CoV-2 VLPs by co-expressing the four SARS-CoV-2 structural proteins, specifically the membrane (M), small envelope (E), spike (S) and nucleocapsid (N) proteins. We show that these proteins are essential and sufficient for the efficient formation and release of SARS-CoV-2 VLPs. Moreover, we used lentiviral vectors to generate human cell lines that stably produce VLPs. Because VLPs can bind to the virus natural receptors, hence leading to entry into cells and viral antigen presentation, this platform could be used to develop novel vaccine candidates that are delivered intranasally.