Analysis of factors that affect pseudotyping in retroviruses

Abstract

Retroviral vectors can efficiently pseudotype with various envelope glycoproteins; however, the molecular mechanism of glycoprotein acquisition process is not completely understood even with their native Env proteins. Understanding this mechanism would aid development of a safe and efficient retroviral gene therapy vector and in finding an antiviral therapeutic target. Various factors were reported to affect the glycoprotein acquisition process. Protein-protein interactions and protein-lipid interactions were reported between viral proteins or cellular factors. In the studies outlined here, we examined three factors that affect retroviral pseudotyping: the HIV-1 accessory protein Vpu, the Env cytoplasmic tail, and cell line-specific host factors. Vpu has been identified to negatively affect Env protein from gibbon ape leukemia virus (GaLV) in a similar way that it down-modulates native targets, CD4 and BST-2/tetherin. We take advantage of a novel CRISPR-Cas9 technique to present here that ßTrCP is a necessary host factor for Vpu to target GaLV Env, CD4, and BST-2/tetherin. The role and structure of Env cytoplasmic tails are largely unknown among retroviral proteins. We present that specific sequences in a gammaretroviral cytoplasmic tail affect fusogenicity in a virus-specific way. It suggests that the lipid environment differs between viruses and that these differences affect fusion of Env. Finally, we present that the cell line specific assembly defect of murine leukemia virus (MLV) particles in HeLa cells. The budding defect of MLV from HeLa cells is restored by fusion with 293FT cells, suggesting that an unknown necessary host factor is deficient in HeLa cells.