Production of Vesicular Stomatitis Virus Glycoprotein-Pseudotyped Lentiviral Vector Is Enhanced by Ezrin Silencing

Mai Izumida; Kei Togawa; Hideki Hayashi; Toshifumi Matsuyama; Yoshinao Kubo

doi:10.3389/fbioe.2020.00368

Production of Vesicular Stomatitis Virus Glycoprotein-Pseudotyped Lentiviral Vector Is Enhanced by Ezrin Silencing

Front Bioeng Biotechnol. 2020 Apr 29:8:368. doi: 10.3389/fbioe.2020.00368. eCollection 2020.

Authors

Mai Izumida^{1

2}, Kei Togawa¹, Hideki Hayashi^{1

3}, Toshifumi Matsuyama^{1

4}, Yoshinao Kubo^{1

2

5}

Affiliations

¹ Department of Molecular Microbiology and Immunology, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan.
² Program for Nurturing Global Leaders in Tropical and Emerging Communicable Diseases, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan.
³ Medical University Research Administrator, Nagasaki University School of Medicine, Nagasaki, Japan.
⁴ Department of Cancer Stem Cell Biology, Institute of Biomedical Sciences, Nagasaki University, Nagasaki, Japan.
⁵ Department of Clinical Medicine, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan.

Abstract

Human immunodeficiency virus type 1 (HIV-1)-based viral vector is widely used as a biomaterial to transfer a gene of interest into target cells in many biological study fields including gene therapy. Vesicular stomatitis virus glycoprotein (VSV-G)-containing HIV-1 vector much more efficiently transduces various mammalian cells than other viral envelope proteins-containing vectors. Understanding the mechanism would contribute to development of a novel method of efficient HIV-1 vector production. HIV-1 vector is generally constructed by transient transfection of human 293T or African green monkey COS7 cells. It was found in this study that HIV-1 Gag protein is constitutively digested in lysosomes of African green monkey cells. Surprisingly, VSV-G elevated HIV-1 Gag protein levels, suggesting that VSV-G protects Gag protein from the lysosomal degradation. Unphosphorylated ezrin, but not phosphorylated ezrin, was detected in COS7 cells, and ezrin silencing elevated Gag protein levels in the presence of VSV-G. Expression of unphosphorylated ezrin reduced Gag protein amounts. These results indicate that unphosphorylated ezrin proteins inhibit the VSV-G-mediated stabilization of HIV-1 Gag protein. Trafficking of HIV-1 Gag-associated intracellular vesicles may be controlled by ezrin. Finally, this study found that ezrin silencing yields higher amount of VSV-G-pseudotyped HIV-1 vector.

Keywords: Gag protein; HIV-1; VSV-G; ezrin; phosphorylation.