Nonhuman Primate IFITM Proteins Are Potent Inhibitors of HIV and SIV

Wilkins, Jordan; Zheng, Yi-Min; Yu, Jingyou; Liang, Chen; Liu, Shan-Lu

SupT1 (NIH AIDS Reagent Program) and Jurkat-inGLuc (gift from Walther Mothes) cells were maintained in Roswell Park Memorial Institute medium (RPMI-1640) with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. HeLa-TZM-bl (NIH AIDS Reagent Program) and HEK293T (ATCC) cells were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% FBS and 1% penicillin-streptomycin. Nonhuman primate fibroblasts were obtained from Coriell Institute cell repositories (Camden, NJ). Fibroblasts were either maintained in DMEM and/or Minimum Essential Medium (MEM) with 10-15% FBS and 1% penicillin-streptomycin according to Coriell's specimen specifications. All cell lines were incubated in a 5% CO2 atmosphere at 37˚C. All IFITMs were cloned into the retroviral vector pQCXIP containing an N-terminal Flag tag. The following proviral DNAs were obtained from NIH AIDS Reagent Program: HIV-1-NL4.3 (#114), HIV-1 BH10 (#90), HIV-2-ROD1 (#207), SIVcpzTAN2.69 (#11497), SIVagm-TAN (#3444), and SIVmac239 (#210). The pCMV-BlaM-Vpr construct was purchased from Addgene (#21950) (described in [43] ). Lentiviral vectors containing control-shRNA or IFITM3-shRNA were purchased from Sigma. The anti-FLAG beads (F2426), anti-FLAG (F3165) and anti-β-tubulin (T8328) antibodies were purchased from Sigma. Anti-IFITM3 antibody was purchased from Proteintech (11714-1-AP). The following antibodies were obtained from NIH AIDS Reagent Program: anti-HIV-1 gp120 (#288), anti-HIV-1 gp41 (#1475), and anti-HIV-1 p24 (#1513).