S1Supplemental Data APOBEC3F Properties and Hypermutation Preferences Indicate Activity against HIV-1 In Vivo

Supplemental Experimental Procedures hr later. Viral stocks were normalized for reverse transcriptase activ-ity by ELISA (Cavidi Tech) and used for infecting 293T cells at 20%– 40 % confluency. Approximately 48 hr after infection, 50,000 targetDNA Constructs Oligonucleotides 5-NNGAGCTCAGGTACCACCATGAAGCCTCACT cells were examined for GFP fluorescence by flow cytometry (FACS-Calibur, Beckton Dickinson). Unless otherwise stated, each experi-TCAGAAAC-3 and 5-NNGTCGACATGAGCAGGAGGCTAGAGGAG-3 were used for amplifying the human APOBEC3F cDNA from IMAGE ment was performed in triplicate, and the mean and standard devia-tion for each data point are shown. Where appropriate, the infectivityclone 6473251 (ATCC). PCR products were digested with Kpn I and Sal I, cloned into pTrc99A (AP Biotech) for expression in E. coli or into resulting from viruses produced with vector controls was used for calculating relative values to facilitate comparison. In all experi-pcDNA3.1 (Invitrogen) for expression in 293T cells, and confirmed by DNA sequencing. The APOBEC3G expression constructs, HIV-1 IIIB ments, 2 g of DNA was transfected. Constant amounts of proviral DNA were used in each experiment, with the APOBEC and Vif plas-Vif expression plasmids (pcVif and pcVif), and MLV-GFP (M3P-GFP) retroviral production system were reported previously [S1, S2]. mids included at the ratios indicated. For MLV-GFP experiments, a plasmid cocktail containing M3P-GFP, pGag/Pol, pMDG (VSV-G),The HIV-GFP retroviral system, including the HIV-1 based proviral plasmid CSCG [S3], pRK5/Pack1(Gag-Pol), pRK5/Rev, and pMDG and the indicated APOBEC expression plasmid (or control) was cotransfected at a ratio of 0.2:0.14:0.06:0.4, respectively. For HIV-(VSV-G Env), was kindly provided by N. Somia (University of Minne