Biology subjects

Elfayres, G.

Publications and source records attributed to Elfayres, G..

3 recordsLinked to original sources

bioRxiv2024

A novel SARS-CoV-2-derived infectious vector system

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19. The development of antiviral drugs for COVID-19 has been hampered by the requirement of a biosafety level 3 (BSL3) laboratory for experiments related to SARS-CoV-2, and by the lack of easy and precise methods for quantification of infection. Here, we developed a SARS-CoV-2 viral vector composed of all four SARS-CoV-2 structural proteins constitutively expressed in lentivirally transduced cells, combined with an RNA replicon deleted for SARS-CoV-2 structural protein genes S, M and E, and expressing a luciferase-GFP fusion protein. We show that, after concentrating viral stocks by ultracentrifugation, the SARS-CoV-2 viral vector is able to infect two human cell lines expressing receptors ACE2 and TMPRSS2. Both luciferase activity and GFP fluorescence were detected, and transduction was remdesivir-sensitive. We also show that this vector is inhibited by three type I interferons (IFN-I) subtypes. Although improvements are needed to increase infectious titers, this vector system may prove useful for antiviral drug screening and SARS-CoV-2-related investigations.

microbiology↗

bioRxiv2024

T7 RNA polymerase-independent expression of reporter genes from a T7 promoter-driven SARS-CoV-2 replicon-encoding DNA in human cells

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. Surprisingly, however, 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.

microbiology↗

bioRxiv2023

Mammalian cells-based platforms for the generation of SARS-CoV-2 virus-like particles

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. HighlightsO_LIIdentification of protein requirements for SARS-CoV-2 VLP production by transient transfection C_LIO_LILentiviral transduction to create cells stably producing SARS-CoV-2 VLPs C_LIO_LIIsolation of cell clones for the production of SARS-CoV-2 VLPs C_LIO_LINew putative platforms for vaccine development C_LI

microbiology↗