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[PMC free content] [PubMed] [CrossRef] [Google Scholar] 6. was possible only when the cells had been built to stably express the individual angiotensin-converting enzyme 2 (ACE2) receptor, but stably presenting an additional duplicate of the receptor didn’t further enhance susceptibility. Steady introduction from the Spike-activating protease TMPRSS2 additional improved susceptibility to infections by 5- to 10-flip. Substitution of the sign peptide from the Spike proteins with an optimum sign peptide didn’t enhance or decrease infectious particle creation. Nevertheless, adjustments D614G and R682Q enhanced infectious particle creation further. With all enhancing elements combined, the titer of pseudotyped HIV-1 particles reached almost 106 infectious particles/ml. Finally, HIV-1 particles pseudotyped with SARS-COV-2 Spike were successfully used to detect neutralizing antibodies in plasma from coronavirus disease 2019 (COVID-19) patients, but not in plasma from uninfected individuals. IMPORTANCE In work with pathogenic viruses, it is useful to have rapid quantitative tests for viral infectivity that can be performed without strict biocontainment restrictions. A common Fissinolide way of accomplishing this is to generate viral pseudoparticles that contain the surface glycoprotein from the pathogenic virus incorporated into a replication-defective viral particle that contains a sensitive reporter system. Fissinolide These pseudoparticles enter cells using the glycoprotein from the pathogenic virus, leading to a readout for infection. Conditions that block entry of the pathogenic virus, such as neutralizing antibodies, will also block entry of the viral pseudoparticles. However, viral glycoproteins often are not readily suited for generating pseudoparticles. Here, we describe a series of modifications that result in the production of relatively high-titer SARS-COV-2 pseudoparticles that are suitable for the detection of neutralizing antibodies from COVID-19 patients. luciferase (Gluc) gene in the reverse orientation containing a forward intron (Fig. 2A) (21). This viral construct produces very low background because it is not capable of producing a luciferase signal unless the gene is reverse transcribed in the target cell. Infectivity with SARS-COV-2 Spike requires expression of the host receptor ACE2. Our starting cell line was generated using a retroviral transfer vector containing ACE2 and a puromycin selection cassette. To determine if introduction of an additional stable copy of ACE2 would enhance susceptibility to infection, we generated a second retroviral transfer vector with a blasticidin resistance cassette. Retroviral particles were generated with this PAX3 vector and used to stably transduce 293FT or 293FT/ACE2 (Puro) cells. Each of the cell lines was transduced with HIV-1-Gluc particles pseudotyped with SARS-COV-2 19 Spike (Fig. 2B). As expected, 293FT cells were not susceptible to infection with the HIV-1/SARS-COV-2 19 Spike. However, both cell lines containing a single introduction of ACE2, and the cell line containing two introductions, had approximately 1,000-fold increases in the luciferase signal, but the surface ACE2 expression and viral susceptibility were roughly equivalent among the three cell lines (Fig. 2B and ?andCC). Open in a separate window FIG 2 Introduction of ACE2 is required for infection of 293FT cells with SARS-COV-2 Spike. (A) Schematic of the HIV-1 luciferase vector. (B) Transduction of 293FT cells transduced with different numbers of ACE2 genes with HIV-1-Gluc pseudoparticles pseudotyped with SARS-COV-2 19 Spike or VSV-G. Transductions with VSV-G used 100 times less viral supernatant. Puro, vector with puromycin selection cassette; Blast, vector with blasticidin resistance cassette. Data are averages and standard errors for three independent experiments. Asterisks indicate significant differences ( 0.05) by a paired Student test; NS, no significant difference. (C) ACE2 surface expression of ACE2-expressing cell lines. Shown is an example representative of the results of three experiments. TMPRSS2 expression enhances susceptibility of cells to SARS-COV-2 pseudotypes. The SARS-COV-2 Spike requires proteolytic priming during infection by either cysteine proteases CatB/L or serine protease TMPRSS2, produced in the target cell (3, 6). To determine whether introduction of TMPRSS2 would enhance susceptibility to infection, we synthesized a codon-optimized TMPRSS2 gene, introduced this gene into a retroviral transfer vector, and stably transduced 293FT or 293FT/ACE2 cells. The stable introduction of TMPRSS2 into 293FT cells did not impart sensitivity to transduction with HIV-1 particles Fissinolide pseudotyped with SARS-COV-2 19 Spike (Fig. 3). However, stable introduction of TMPRSS2 into 293FT/ACE2 cells increased the Gluc signal from target cells by 5- to 10-fold. Neither ACE2 expression nor TMPRSS2 expression affected the Gluc signal from particles pseudotyped with VSV-G. Open in a separate window FIG 3 TMPRSS2 expression enhances susceptibility to infection with SARS-COV-2 Spike. 293FT cells stably expressing ACE2, TMPRSS2, or both were transduced with HIV-1-Gluc particles pseudotyped with SARS-COV-2 19 Spike or VSV-G. Transductions with VSV-G used.