key: cord-0826159-4t6f7wul authors: Johnson, Marc C.; Lyddon, Terri D.; Suarez, Reinier; Salcedo, Braxton; LePique, Mary; Graham, Maddie; Ricana, Clifton; Robinson, Carolyn; Ritter, Detlef G. title: Optimized pseudotyping conditions for the SARS-COV2 Spike glycoprotein date: 2020-05-29 journal: bioRxiv DOI: 10.1101/2020.05.28.122671 sha: 044d1188e9e5dfde6446b2d069626eb9137d0d1c doc_id: 826159 cord_uid: 4t6f7wul The SARS-COV2 Spike glycoprotein is solely responsible for binding to the host cell receptor and facilitating fusion between the viral and host membranes. The ability to generate viral particles pseudotyped with SARS-COV2 Spike is useful for many types of studies, such as characterization of neutralizing antibodies or development of fusion-inhibiting small molecules. Here we characterized the use of a codon-optimized SARS-COV2 Spike glycoprotein for the generation of pseudotyped HIV-1, MLV, and VSV particles. The full-length Spike protein functioned inefficiently with all three systems but was enhanced over 10-fold by deleting the last 19 amino acids of the cytoplasmic tail of Spike. Infection of 293FT target cells was only possible if the cells were engineered to stably express the human ACE-2 receptor, but stably introducing an additional copy of this receptor did not further enhance susceptibility. Stable introduction of the Spike activating protease TMPRSS2 further enhanced susceptibility to infection by 5-10 fold. Substitution of the signal peptide of the Spike protein with an optimal signal peptide did not enhance or reduce infectious particle production. However, modification of a single amino acid in the furin cleavage site of Spike (R682Q) enhanced infectious particle production another 10-fold. With all enhancing elements combined, the titer of pseudotyped particles reached almost 106 infectious particles/ml. Finally, HIV-1 particles pseudotyped with SARS-COV2 Spike was successfully used to detect neutralizing antibodies in plasma from COVID-19 patients, but not plasma from uninfected individuals. Importance When working with pathogenic viruses, it is useful to have rapid quantitative tests for viral infectivity that can be performed without strict biocontainment restrictions. A common 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. 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-COV2 pseudoparticles that are suitable for detection of neutralizing antibodies from COVID-19 patients. 19 amino acids of the cytoplasmic tail of Spike. Infection of 293FT target cells was only 22 possible if the cells were engineered to stably express the human ACE-2 receptor, but stably 23 introducing an additional copy of this receptor did not further enhance susceptibility. Stable 24 introduction of the Spike activating protease TMPRSS2 further enhanced susceptibility to 25 infection by 5-10 fold. Substitution of the signal peptide of the Spike protein with an optimal 26 signal peptide did not enhance or reduce infectious particle production. However, modification of 27 a single amino acid in the furin cleavage site of Spike (R682Q) enhanced infectious particle 28 production another 10-fold. With all enhancing elements combined, the titer of pseudotyped 29 particles reached almost 10 6 infectious particles/ml. Finally, HIV-1 particles pseudotyped with 30 SARS-COV2 Spike was successfully used to detect neutralizing antibodies in plasma from 31 COVID-19 patients, but not plasma from uninfected individuals. Importance. When working with pathogenic viruses, it is useful to have rapid quantitative tests for viral 35 infectivity that can be performed without strict biocontainment restrictions. A common way of 36 accomplishing this is to generate viral pseudoparticles that contain the surface glycoprotein from 37 the pathogenic virus incorporated into a replication-defective viral particle that contains a 38 sensitive reporter system. These pseudoparticles enter cells using the glycoprotein from the pathogenic virus leading to a readout for infection. Conditions that block entry of the pathogenic 40 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 42 we describe a series of modifications that result in the production of relatively high titer SARS-43 COV2 pseudoparticles that are suitable for detection of neutralizing antibodies from COVID-19 44 patients. The COVID-19 pandemic is a severe threat to human health and the global economy. COVID-52 19 is caused by infection with the SARS-CoV-2 virus, which is a highly pathogenic 53 betacoronavirus (1, 2). A critical tool for the study of pathogenic viruses such as SARS-COV2 is 54 a rapid and sensitive assay for agents that block viral entry such as neutralizing antibodies or 55 small molecule inhibitors. A safe and powerful technique for generating such an assay is to 56 generate viral pseudotyped particles where the surface fusion protein of the pathogen of interest 57 is assembled onto the surface of replication-defective virus which contains a sensitive reporter 58 protein. The Spike glycoprotein from Coronaviruses facilitates binding to the host cell receptor and 61 fusion between viral and cellular membranes. The SARS-COV2 Spike protein is a large 1,274 62 amino acid protein that contains an N-terminal S1 receptor binding domain and a C-terminal S2 63 fusion domain. Fusion by SARS-COV2 Spike requires binding to the host receptor ACE2 (3-6) 64 as well as proteolytic cleavage by host proteases at the S1/S2 and S2' positions by host cysteine proteases cathepsin B and L (CatB/L) or serine protease TMPRSS2 (3, 6). Depending 66 on the cell line, inhibition of one or both of these proteases is sufficient to block viral entry. An 67 interesting difference between the SARS-COV1 and COV2 Spike proteins is the presence of a 68 furin cleavage site near the S1/S2 cleavage site (6, 7). This cleavage site was found to be 69 essential for infection of human lung cells (3). Surprisingly, passaging of SARS-COV2 in Vero 70 E6 cells selects for mutations that alter the furin cleavage site, resulting in virus that produce 71 larger plaque sizes (8). There have been several reports generating SARS-COV1 (9-12) and 13, 14) 74 viral pseudotypes with glycoprotein-defective MLV, HIV, and VSV particles. Although engineered to express human ACE2. Infectious particle production with all three types of 95 particles was very low with the full-length Spike protein, but the 19 Spike produced viral titers 96 approaching 10 4 infectious particles/ml with both HIV-1 and VSV particles. The infectious 97 particle production remained over 1000-fold lower than with the control glycoprotein, VSV-G. To allow rapid quantitation with minimum background, we proceeded with an Env- 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-117 optimized TMPRSS2 gene, introduced this gene into a retroviral transfer vector, and stably 118 transduced 293FT or 293FT/ACE2 cells. The stable introduction of TMPRSS2 to 293FT cells 119 did not impart sensitivity to transduction with HIV-1 particles pseudotyped with SARS-COV2 19 120 Spike (Fig. 3) . However, stable introduction of TMPRSS2 to 293FT/ACE2 cells increased the 121 Gluc signal from target cells by 5-10 fold. Neither ACE2 expression nor TMPRSS2 expression 122 affected Gluc signal from particles pseudotyped with VSV-G. The R682Q mutation in SARS-COV2 Spike enhances pseudotyping efficiency. To explore 124 additional genetic modifications in SARS-COV2 that might enhance infectious particle 125 production, we replaced the endogenous signal peptide with a well characterized strong signal 126 peptide (16), and separately we introduced a point mutation in the furin-cleavage site of SARS-127 COV2 19 Spike (R682Q) that has previously been reported to increase SARS-COV2 plaque 128 sizes ( Fig. 4 (8) ). Infectious particle production was assessed using both Gluc and Cre 129 reporters, and transductions were performed in cell lines with and without TMPRSS2. To 130 minimize target cell variation, the transductions were all performed in 293FT/Cre-reporter/ACE2 131 and 293FT/Cre-reporter/ACE2/TMPRSS2 cells. Replacement of the signal peptide did not 132 increase or decrease infectious particle production ( Fig 4A) . However, the R682Q mutation 133 significantly enhanced infectious particle production under all conditions. The enhancement 134 was most pronounced with virus containing the Cre reporter in cells lacking TMPRSS2 (11-fold). The highest titer of 8x10 5 was achieved with the R682Q 19 Spike into cells expressing both 136 ACE2 and TMPRSS2. Discussion. Here we outline glycoprotein and target cell modifications that enhance pseudotyping 229 efficiency with the SARS-COV2 Spike glycoprotein. The first and most important changes are 230 the necessity to express the human ACE2 receptor, which has been defined previously by 231 numerous investigators, and the enhancement gained from truncation of the Spike cytoplasmic 232 tail. Truncation of the last 19 amino acids for SARS-COV1 was also shown to significantly 233 enhance pseudotyping with that glycoprotein (9). In that study, the original reason for making 234 the truncation was to eliminate potential ER retention sequences in the cytoplasmic tail. Despite 235 enhancing pseudotyping capacity, the truncation did not enhance surface expression. It is likely 236 that the enhancement in pseudotyping efficiency upon deletion of the cytoplasmic is at least in 237 part due to eliminating steric interference of the cytoplasmic tail with the viral capsid. Such 238 interference has been noted when pseudotyping other viral glycoproteins with large cytoplasmic 239 tails, such as HIV-1 Env (23, 24). The next modification that enhanced efficiency was the introduction of the human 241 protease TMPRSS2. The importance of this protease for SARS-COV2 Spike glycoprotein entry 242 has been noted previously, so this enhancement was not surprising (3, 6). The final modification that enhanced SARS-COV2 19 Spike pseudotyping was the 244 introduction of the R682Q mutation, which is predicted to eliminate the furin cleavage site. This A possible explanation is that furin cleavage enhances TMPRSS2 mediated entry, but 253 suppresses CatB/L mediated entry. Consistent with this, the enhancement we observed from 254 the R682Q mutation was less pronounced in cells that express TMPRSS2. It should be noted Finally, we demonstrate that HIV-1-Gluc particles pseudotyped with SARS-COV2 19 260 Spike could be used for detecting neutralizing antibodies from COVID-19 patient plasma. Inclusion of the R682Q in Spike did not obviously change the neutralization results in this assay. Thus, this appears to be a suitable system for studying inhibitors of SARS-COV2 entry. China Novel Coronavirus I, Research T. 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