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

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

doi:10.3389/fbioe.2020.00368

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.

Highlights

A lentiviral vector based on human immunodeficiency virus type 1 (HIV-1) is widely used as a vector to transfer a gene of interest into target cells in many biological study fields including basic biology and clinical gene therapy
To assess whether viral envelope glycoproteins stabilize Human immunodeficiency virus type 1 (HIV-1) Gag protein, African green monkey COS7 cells were transfected with the HIV-1 Gag-Pol expression plasmid lacking the packaging signal together with pcDNA3.1, HXB2 HIV-1 Envelope proteins (Envs), or Vesicular stomatitis virus glycoprotein (VSV-G) expression plasmid
This result indicates that VSV-G elevates HIV-1 Gag protein amount, but HIV-1 Env protein does not

Summary

Introduction

A lentiviral vector based on human immunodeficiency virus type 1 (HIV-1) is widely used as a vector to transfer a gene of interest into target cells in many biological study fields including basic biology and clinical gene therapy. 2019), because these cells express the simian virus 40 T antigen that induces DNA replication of the SV40 replication origin-containing plasmids in transfected cells. Host cells usually contain restriction mechanisms to inhibit viral replication (Ghimire et al, 2018). Such host restriction mechanisms prevent production of lentiviral vector. Accessory proteins encoded by HIV-1 induce degradation of the host restriction factors to escape from host innate immunity. Lentivirus vector generally do not express the accessory proteins to prevent unexpected effects of those viral proteins. There is no method for inactivation of host restriction factors to produce high amount of HIV-1 vector particles

Methods

Results

Conclusion