Table_1_Production of Vesicular Stomatitis Virus Glycoprotein-Pseudotyped Lentiviral Vector Is Enhanced by Ezrin Silencing.DOCX

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.

人免疫缺陷病毒1型（HIV-1）病毒载体被广泛用作生物材料，在包括基因治疗在内的众多生物学研究领域中，将感兴趣的基因转移至靶细胞。含有囊性纤维化病毒糖蛋白（VSV-G）的HIV-1载体比其他含有病毒包膜蛋白的载体更有效地转导各种哺乳动物细胞。理解其机制将有助于开发一种高效HIV-1病毒载体生产的新方法。HIV-1病毒载体通常通过瞬时转染人293T或非洲绿猴COS7细胞构建。本研究发现，HIV-1 Gag蛋白在非洲绿猴细胞溶酶体中持续被消化。令人惊讶的是，VSV-G提高了HIV-1 Gag蛋白的水平，这表明VSV-G能够保护Gag蛋白免受溶酶体降解。在COS7细胞中检测到非磷酸化ezrin，而非磷酸化ezrin的沉默在VSV-G存在下提高了Gag蛋白水平。非磷酸化ezrin的表达减少了Gag蛋白的数量。这些结果表明，非磷酸化ezrin蛋白抑制了VSV-G介导的HIV-1 Gag蛋白的稳定化。与HIV-1 Gag蛋白相关的细胞内囊泡的运输可能受到ezrin的调控。最终，本研究发现，ezrin的沉默产生了更高数量的VSV-G伪型化的HIV-1病毒载体。