Citation
- Authors: Ganaie SS. et al.
- Year: 2021
- Journal: Cell 184 5163-5178
- Applications: in vitro / DNA / FectoPRO
- Cell type: Expi293F
Description: Human embryonic kidney Fibroblast
Known as: Expi 293-F, Expi, HEK-293 Expi
Method
IgG production.
DNA encoding the variable regions of phage-derived antibodies was amplified from phagemid DNA by the PCR and sub-cloned in to separate light and heavy chain expression vectors. Equal amounts of DNA from heavy and light chain expression vectors were mixed, diluted in Opti-MEM medium (Gibco), and complexed with FectoPro transfection reagent (Polyplus Transfection) for 10 minutes. Complexed DNA was transfected in to Expi-293F cells in Expi293 medium and and the cultures were incubated for 5 days at 37 °C in a humidified, 8% CO2 environment with shaking. Secreted IgG protein was purified from supernatants with Protein A sepharose (GE Healthcare), eluted in IgG elution buffer (Thermo), neutralized with 1 M Tris buffer pH 8.0 (Invitrogen), and exchanged in to PBS using centrifugal concentrators.
Abstract
Rift Valley fever virus (RVFV) is a zoonotic pathogen with pandemic potential. RVFV entry is mediated by the viral glycoprotein (Gn), but host entry factors remain poorly defined. Our genome-wide CRISPR screen identified low-density lipoprotein receptor-related protein 1 (mouse Lrp1/human LRP1), heat shock protein (Grp94), and receptor-associated protein (RAP) as critical host factors for RVFV infection. RVFV Gn directly binds to specific Lrp1 clusters and is glycosylation independent. Exogenous addition of murine RAP domain 3 (mRAPD3) and anti-Lrp1 antibodies neutralizes RVFV infection in taxonomically diverse cell lines. Mice treated with mRAPD3 and infected with pathogenic RVFV are protected from disease and death. A mutant mRAPD3 that binds Lrp1 weakly failed to protect from RVFV infection. Together, these data support Lrp1 as a host entry factor for RVFV infection and define a new target to limit RVFV infections.