Fig. 4.

IFIH1 (MDA5) protein is required for ISG15-induction by HIV-1 in myeloid cells. (A) Schematic representation of all-in-one shRNA-rescue lentivector, in which the SFFV promoter expresses a tripartite fusion of puromycin N-acetyltransferase (puroR), the K48R mutant of ubiquitin (UbK48R), and an ORF encoding the gene of interest, as well as a modified miR30-based shRNA (miR-30). (B–D) All-in-one lentivectors encoding HSA/CD24, or nontargetable, shRNA-resistant IFIH1 coding sequence (ntIFIH1), along with shRNA targeting Luc or IFIH1 as indicated in A, were used to transduce dendritic cells (B and C), or macrophages (D). (B) Representative immunoblot for quantification of IFIH1 (MDA5) protein levels in indicated samples. (C and D) The percentage of ISG15⁺ cells was quantified by flow cytometry 3 d after HIV-1-GFP transduction and normalized to 100% for HSA ORF/Luc-knockdown cells (mean ± SEM, n = 4 donors for each). P values were determined by one-way ANOVA with Tukey’s multiple comparisons test.