Because short-hairpin RNAs against IFITMs (shRNA IFITMs) exhibit cross-knockdown activities against different IFITM orthologs [6], making the evaluation of the contributions of individual IFITMs to HIV-1 entry challenging, we decided to knock down all three IFITMs altogether by using a mixed pool of shRNAs and compared the overall function of IFITMs on infection of CXCR4 and CCR5 HIV-1 strains, i.e., NL4.3 and AD8, respectively. Here, CXCR4 is linked to infection.