Optimized Pseudotyping Conditions for the SARS-COV-2 Spike Glycoprotein

Abstract

The SARS COV-2 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 COV-2 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 COV-2 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, modifications D614G and R682Q further enhanced infectious particle production. 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 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 COV-2 pseudoparticles that are suitable for detection of neutralizing antibodies from COVID-19 patients.

Spike glycoprotein and host cell determinants of SARS-CoV-2 entry and cytopathic effects

“…Although the VSV(S) pseudotypes exhibited higher infectivity than HIV-1(S) pseudotypes, infection of 293T-ACE2 cells by both S-pseudotyped vectors was at least 60-fold above the background seen for viruses without envelope glycoproteins (data not shown). Consistent with previous findings (38,61), expression of TMPRSS2 in the 293T-ACE2 target cells increased the efficiency of virus infection mediated by the wild-type SARS-CoV-2 S gp (Fig. 7A).…”

SARS-CoV-2 Spike Alterations Enhance Pseudoparticle Titers and Replication-Competent VSV-SARS-CoV-2 Virus

“…In various pseudotyped particle and viral particle assays, there appears to be little if any increase in viral titers with S-G614 in tissue culture settings [37,[58][59][60]. Here, the addition of D614G to S Met1 ∆21 did not significantly enhance S incorporation onto pseudoparticles or transduction efficiency beyond the enhancement from truncating the cytoplasmic tail.…”

Efficient production of Moloney murine leukemia virus-like particles pseudotyped with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein

“…7B). The hypothesis that has been proposed for SARS-COV and SARS-CoV-2 is that the 19 amino-acid deletion in the S cytoplasmic tail facilitates the pseudotyping by decreasing the steric interference with the retroviral matrix proteins (54,55,57). Our results invalidate this hypothesis as more DS was also found in the supernatant of 293 transfected cells that did not express MLV Gag-Pol (Fig.…”

“…Mutations of the furin cleavage site located between S1 and S2 and the D614G variant that is now more prevalent in the infected population could increase the amount of S incorporated into VLPs (57,62).…”