Postal address Krišjāņa Barona iela 14 - 29, Rīga, LV-1050 Rīga, Krišjāņa Barona iela 14 - 29, LV-1050 Previous addresses Other registered in this address These cells have been deposited in the repository of the National Institute for Biological Standards and Control’s Covid-19-related research reagents (CFAR #101008) for widespread use.Extended analysis of the company 11.88 EUR This protocol has been recently used to produce lentiviral vectors pseudotyped with different SARS-CoV-2’s Spike variants. Produced vectors are then quantified by an infectivity assay on genetically engineered HEK293T/17 cells that stably express hACE2 and TMPRSS2 (Supplementary Figure 1). ), under the cytomegalovirus (CMV) promoter with its ER retention signal (amino acid residues 1255 to 1273) deleted as it has been shown to enhance surface expression ( The protocol employs a three-plasmid transfection in HEK293T/17 cells with the following plasmids: (i) plasmid expressing the HIV-1 lentiviral genes gag, pol, rev, and tat: (ii) a self-inactivating transfer vector encoding eGFP under an internal viral promoter derived from the spleen focus-forming virus (SFFV) and (iii) a plasmid encoding codon-optimized Spike, with or without the prevalent D614G mutation ( Herein, we describe a fast and reliable protocol for the production of a self-inactivating lentiviral vector pseudotyped with SARS-CoV-2’s Spike glycoprotein and expressing enhanced green fluorescent protein (eGFP) as a marker of infection, which has recently been used to determine neutralisation efficiency of COVID-19 therapeutics on four SARS-CoV-2 variants ( However, a detailed protocol of pseudovector production has not been described for widespread application. ) based vectors for neutralization assays, which have been reported to correlate with the live strain. Accordingly, different viral vectors have been pseudotyped with Spike from SARS-CoV-2, including VSV ( Spike from different coronaviruses have been successfully pseudotyped on different non-replication competent viruses ( Thus, Spike has been shown to be the primary target for neutralizing antibodies in COVID-19 convalescent patient sera ( Spike is both necessary and sufficient to induce membrane fusion and cell entry by first binding to its human receptor, ACE2 (hACE2), followed by its proteolytic cleavage by target cell proteases such as the transmembrane protease serine 2 (TMPRSS2) ( In host cells, precursor glycoproteins are proteolytically cleaved by furin at the multibasic S1/S2 site, resulting in dimers composed of an extracellular subunit (S1) containing the receptor-binding domain, which is non-covalently attached to a transmembrane subunit (S2) responsible for viral fusion and subsequent cell entry ( Spike is a type-I fusion transmembrane protein expressed on the surface of viral particles as a crown-shaped trimer of heterodimers. This allows the deficient core to be dependent on the pseudotyping envelope for target cell entry, thus allowing the investigation of SARS-CoV-2 infection and related serological responses. Pseudotyped viral particles consist of the envelope glycoprotein of one virus with a replication-deficient core of another virus. Pseudotyped viral vectors are very powerful tools for studying biological processes related to enveloped viruses, such as viral entry and immunological response. An alternative to using live virus is to use recombinant lentivirus pseudotyped with the SARS-CoV-2 Spike protein. Studies on SARS-CoV-2 viruses are hampered by the difficulty to produce and manipulate the live viruses that require biosafety level 3 (BSL-3) labs. Production and quantification of lentiviral vectors pseudotyped with the SARS-CoV-2 Spike glycoprotein For the widespread use of this protocol, its reagents have been made publicly available. Enhanced and consistent cell entry is achieved by using permissive HEK293T/17 cells that were genetically engineered to stably express the SARS-CoV-2 human receptor ACE2 along with the cell surface protease TMPRSS2 required for efficient fusion. High titers (>1.00 E+06 infectious units/ml) are produced using codon-optimized CoV-2 S, harbouring the prevalent D614G mutation and lacking its ER retention signal. The virus titer is determined by the GFP reporter (fluorescent) expression with a flow cytometer. Herein, we describe a fast and reliable protocol for the transient production of lentiviruses pseudotyped with SARS-CoV-2 Spike (CoV-2 S) proteins and green fluorescent protein (GFP) reporters. The use of recombinant lentivirus pseudotyped with the coronavirus Spike protein of SARS-CoV-2 would circumvent the requirement of biosafety-level 3 (BSL-3) containment facilities for the handling of SARS-CoV-2 viruses.
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