Robust Enhancement of Lentivirus Production by Promoter Activation

Naoto Suzuki,Sayaka Sukegawa,Takeshi Yoshida,Hiroaki Takeuchi,Ryuta Sakuma,Shoji Yamaoka

doi:10.1038/s41598-018-33042-5

Abstract

Lentiviral vectors are a valuable tool to deliver exogenous genes for stable expression in cells. While much progress has been made in processing lentiviral vector-containing culture medium, it remains to be explored how the production of lentiviral vector from producer cells can be increased. We initially found that co-expression of the SPRY domain-containing SOCS box protein 1 (SPSB1) promotes the production of human immunodeficiency virus type 1 (HIV-1) and lentiviral vector with increased expression of the Gag and envelope proteins and activation of the HIV-1 LTR and CMV promoter. The presence of AP-1, NF-κB and CREB/ATF recognition sites in these promoters prompted us to utilize human T-lymphotropic virus type 1 (HTLV-1) Tax for lentiviral vector production because Tax activates all these transcription factors. Co-expression of a small amount of Tax markedly increased both the expression of viral structural proteins in producer cells and release of lentiviral vector particles, resulting in a more than 10-fold enhancement of transduction efficiency. Of note, the Tax protein was not detected in the lentiviral vector particles concentrated by ultracentrifugation, supporting the safety of this preparation. Collectively, these results indicate that promoter activation in producer cells represents a promising approach to preparing high-titer lentiviral vectors.

Highlights

Lentiviral vectors derived from human immunodeficiency virus type 1 (HIV-1) are a valuable tool to transduce dividing and non-dividing cells with exogenous genes both in vitro and in vivo
To assess whether SPRY domain-containing SOCS box protein 1 (SPSB1) increased virus release from producer cells, we measured the reverse transcriptase (RT) activity in the culture supernatants, which was elevated in the presence of SPSB1 (Fig. 1c)
Quantification of western blotting signal confirmed concordant increase in Gag and Vesicular stomatitis virus glycoprotein (VSV-G) protein expression in producer cells as well as elevated incorporation of VSV-G protein into virus particles (Supplementary Fig. S1). These results indicate that ectopic expression of SPSB1 increased the expression of viral structural proteins in producer cells and suggest that this led to enhanced lentivirus production

Summary

Introduction

Lentiviral vectors derived from human immunodeficiency virus type 1 (HIV-1) are a valuable tool to transduce dividing and non-dividing cells with exogenous genes both in vitro and in vivo. In vivo experiments and transduction of non-dividing cells often require a high-titer of the lentiviral vector, which has to be manufactured in large-scale cultures, concentrated by ultracentrifugation, and purified by chromatography and ultrafiltration[4]. This procedure currently represents a major obstacle because of the high cost for vector preparation using large quantities of plasmids and cell culture materials as well as equipment for concentration and purification. We demonstrate that both SPSB1 and HTLV-1 Tax enhance CMV promoter-driven gene expression in HEK293T cells and have exploited this finding for lentiviral vector production

Methods

Results

Conclusion