LDLRAD3 Mutational Analysis for Viral Infection

A comprehensive mutation library was generated using gene synthesis by mutating a single amino acid in D1 of the LDLRAD3 protein. The amino acids that are essential for maintaining the structural integrity of LDLRAD3 (the cysteines forming disulfide bonds, the amino acids coordinating the calcium and those forming the hydrophobic core) were kept intact^{39 (link)}. The substitutions were determined using the BLOSUM scoring matrix^{40 (link)} and a list of these is provided in Supplementary Table 4. The mutants were cloned into lentivirus vector pLV-EF1a-IRES-Hygro (Addgene, 85134) between the BamHI and MluI restriction enzyme sites (Genscript). An N-terminal Flag tag was added to each LDLRAD3 mutant to monitor protein expression. ∆B4galt7∆Ldlrad3 Neuro2a cells were transduced with each LDLRAD3 mutant and, 7 d later, were inoculated with SINV–VEEV (TrD)–GFP^{1 (link)} (gift of W. Klimstra, University of Pittsburgh) infection at a multiplicity of infection of 20 for 7.5 h. Cells were stained with anti-Flag antibodies (1:2,000 dilution, Cell Signaling Technology, D6W5B) to measure the surface expression levels of the WT and mutant forms of LDLRAD3. Inoculated and stained cells were analysed using the MACSQuant Analyzer 10 (Miltenyi Biotec), and all flow cytometry data were processed using FlowJo (FlowJo).