4.6 Article

Cytoplasmic Tail Truncation of SARS-CoV-2 Spike Protein Enhances Titer of Pseudotyped Vectors but Masks the Effect of the D614G Mutation

期刊

JOURNAL OF VIROLOGY
卷 95, 期 22, 页码 -

出版社

AMER SOC MICROBIOLOGY
DOI: 10.1128/JVI.00966-21

关键词

D614G mutation; SARS-CoV-2; Spike protein; cytoplasmic tail truncation; pseudotyped vectors

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资金

  1. W. M. Keck Foundation COVID-19 Research Fund
  2. Keck School of Medicine of USC

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The high pathogenicity of SARS-CoV-2 requires handling under biosafety level 3 conditions. Spike protein-pseudotyped vectors are useful for studying viral entry and inhibition, with methods to increase titer commonly including ultracentrifugation and Spike protein tail truncation. Studies showed that truncating the Spike tail increased incorporation into vectors and enhanced titers without impacting sensitivity to neutralization, while the D614G mutation independently increased incorporation and titers but was masked by the truncation. Use of full-length Spike protein with tangential flow filtration is recommended for generating high titer pseudotyped vectors retaining native Spike protein functions.
The high pathogenicity of SARS-CoV-2 requires it to be handled under biosafety level 3 conditions. Consequently, Spike protein-pseudotyped vectors are a useful tool to study viral entry and its inhibition, with retroviral, lentiviral (LV), and vesicular stomatitis virus (VSV) vectors the most commonly used systems. Methods to increase the titer of such vectors commonly include concentration by ultracentrifugation and truncation of the Spike protein cytoplasmic tail. However, limited studies have examined whether such a modification also impacts the protein's function. Here, we optimized concentration methods for SARS-CoV-2 Spike-pseudotyped VSV vectors, finding that tangential flow filtration produced vectors with more consistent titers than ultracentrifugation. We also examined the impact of Spike tail truncation on transduction of various cell types and sensitivity to convalescent serum neutralization. We found that tail truncation increased Spike incorporation into both LV and VSV vectors and resulted in enhanced titers but had no impact on sensitivity to convalescent serum. In addition, we analyzed the effect of the D614G mutation, which became a dominant SARS-CoV-2 variant early in the pandemic. Our studies revealed that, similar to the tail truncation, D614G independently increases Spike incorporation and vector titers, but this effect is masked by also including the cytoplasmic tail truncation. Therefore, the use of full-length Spike protein, combined with tangential flow filtration, is recommended as a method to generate high titer pseudotyped vectors that retain native Spike protein functions. IMPORTANCE Pseudotyped viral vectors are useful tools to study the properties of viral fusion proteins, especially those from highly pathogenic viruses. The Spike protein of SARS-CoV-2 has been investigated using pseudotyped lentiviral and VSV vector systems, where truncation of its cytoplasmic tail is commonly used to enhance Spike incorporation into vectors and to increase the titers of the resulting vectors. However, our studies have shown that such effects can also mask the phenotype of the D614G mutation in the ectodomain of the protein, which was a dominant variant arising early in the COVID-19 pandemic. To better ensure the authenticity of Spike protein phenotypes when using pseudotyped vectors, we recommend using full-length Spike proteins, combined with tangential flow filtration methods of concentration if higher-titer vectors are required.

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