Cytoplasmic tail truncation of SARS-CoV-2 Spike protein enhances titer of pseudotyped vectors but masks the effect of the D614G mutation

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 inhibition. 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 that 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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Production of high‐titer transmission‐defective RNA virus‐based episomal vector using tangential flow filtration

Microbiology and Immunology ◽

10.1111/1348-0421.12831 ◽

2020 ◽

Vol 64 (9) ◽

pp. 602-609

Author(s):

Yumiko Komatsu ◽

Yoji Kakuya ◽

Keizo Tomonaga

Keyword(s):

Rna Virus ◽

High Titer ◽

Tangential Flow Filtration ◽

Episomal Vector ◽

Tangential Flow ◽

Defective Rna ◽

Flow Filtration

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Preparation of pure, high titer, pseudoinfectious Flavivirus particles by hollow fiber tangential flow filtration and anion exchange chromatography

Vaccine ◽

10.1016/j.vaccine.2014.09.074 ◽

2015 ◽

Vol 33 (35) ◽

pp. 4255-4260 ◽

Cited By ~ 7

Author(s):

Sophia T. Mundle ◽

Maryann Giel-Moloney ◽

Harry Kleanthous ◽

Konstantin V. Pugachev ◽

Stephen F. Anderson

Keyword(s):

Anion Exchange ◽

Hollow Fiber ◽

High Titer ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Tangential Flow Filtration ◽

Tangential Flow ◽

Flow Filtration

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The Effects of Aβ1-42 Binding to the SARS-CoV-2 Spike Protein S1 Subunit and Angiotensin-Converting Enzyme 2

International Journal of Molecular Sciences ◽

10.3390/ijms22158226 ◽

2021 ◽

Vol 22 (15) ◽

pp. 8226

Author(s):

John Tsu-An Hsu ◽

Chih-Feng Tien ◽

Guann-Yi Yu ◽

Santai Shen ◽

Yi-Hsuan Lee ◽

...

Keyword(s):

Angiotensin Converting Enzyme ◽

Viral Entry ◽

Negative Impact ◽

Spike Protein ◽

Infection Model ◽

Converting Enzyme ◽

Viral Receptor ◽

Angiotensin Converting Enzyme 2 ◽

People With Dementia ◽

The Impact

Increasing evidence suggests that elderly people with dementia are vulnerable to the development of severe coronavirus disease 2019 (COVID-19). In Alzheimer’s disease (AD), the major form of dementia, β-amyloid (Aβ) levels in the blood are increased; however, the impact of elevated Aβ levels on the progression of COVID-19 remains largely unknown. Here, our findings demonstrate that Aβ1-42, but not Aβ1-40, bound to various viral proteins with a preferentially high affinity for the spike protein S1 subunit (S1) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the viral receptor, angiotensin-converting enzyme 2 (ACE2). These bindings were mainly through the C-terminal residues of Aβ1-42. Furthermore, Aβ1-42 strengthened the binding of the S1 of SARS-CoV-2 to ACE2 and increased the viral entry and production of IL-6 in a SARS-CoV-2 pseudovirus infection model. Intriguingly, data from a surrogate mouse model with intravenous inoculation of Aβ1-42 show that the clearance of Aβ1-42 in the blood was dampened in the presence of the extracellular domain of the spike protein trimers of SARS-CoV-2, whose effects can be prevented by a novel anti-Aβ antibody. In conclusion, these findings suggest that the binding of Aβ1-42 to the S1 of SARS-CoV-2 and ACE2 may have a negative impact on the course and severity of SARS-CoV-2 infection. Further investigations are warranted to elucidate the underlying mechanisms and examine whether reducing the level of Aβ1-42 in the blood is beneficial to the fight against COVID-19 and AD.

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