The full-length molecular HIV-1 clone LAI (29 (link)) was used to produce wild-type and mutant viruses. Nucleotide numbers presented here refer to the position on the genomic HIV-1 RNA transcript, with +1 being the capped G residue. The mutant proviral DNA sequences were PCR-amplified from cellular DNA with the 5′ Env primer tTA1-AD (+8269 to +8289) and the 3′ U5 primer CN1 (+9253 to +9283). The PCR fragments were digested with XhoI and BspEI, and cloned into the plasmid Blue-3′LTR (30 (link)). The XhoI–BglI fragments (1709 bp) of these plasmids were cloned into the wild-type LAI clone, resulting in the full-length mutant clones R1–R9.
The firefly luciferase expression vector pGL3 control (Promega) was used to construct the wild-type and mutant reporter-Nef target plasmids (pGL3-Nef). An approximately 250 bp Nef fragment (+8448 to +8698) was PCR amplified from the full-length molecular clones with the primers EW1 (5′-ACGTCTAGAATTCTGAGACGAGCTGAGCCAGCA-3′) and EW3 (5′-GACTCTAGACTGCAGGAGTGAATTAGCCCTTCCA-3′). The PCR product was digested with XbaI and cloned into the XbaI site located downstream of the luciferase gene in pGL3 control. The forward orientation of the insert was checked by sequence analysis.
To construct the m1–m4 mutants, base changes were introduced into pGL3-Nef by mutagenesis PCR (31 (link)). Mutagenic (m) primers EWmut1 (5′-ACAGCAGCTACCAATC CTGCTTGTGC -3′, mismatching nucleotide underlined; m1), EWmut2 (5′-CACAAGTAGG AATACAGCAGCTACCAAC CTGCTTGTGC-3′; m2), EWmut3 (5′-CACAAGTAGT AATACAGCAG-3′; m3 and m4), and the general primers EW1 (Primer 1), EW2 (5′-TGAGGCCCGGTACCTGAGGTGTGACT-3′; primer 2), and EW3 (Primer 3) were used with the wild-type pGL3-Nef (m1, m2 and m4) or the R8 pGL3-Nef (m3) template. Briefly, PCR reactions were performed with primer M plus primer 3, and with primer 1 plus primer 2. The PCR products were purified, mixed and PCR amplified with primers 1 and 3 as described previously (31 (link)). The PCR fragments were digested with XbaI and cloned in the corresponding site of pGL3 control. All mutations were verified by sequence analysis.
The pRetro-SUPER-shNef vector, which expresses siRNA-Nef under control of the H1 RNA polymerase III promoter was digested with EcoRI and XhoI, and the 314 bp expression cassette was ligated into the EcoRI/XhoI sites of pBluescriptII (KS+) (Stratagene) to produce pBS-siRNA-Nef. Plasmid pRL-CMV (Promega) expresses renilla luciferase under control of the CMV promoter.
The firefly luciferase expression vector pGL3 control (Promega) was used to construct the wild-type and mutant reporter-Nef target plasmids (pGL3-Nef). An approximately 250 bp Nef fragment (+8448 to +8698) was PCR amplified from the full-length molecular clones with the primers EW1 (5′-ACGTCTAGAATTCTGAGACGAGCTGAGCCAGCA-3′) and EW3 (5′-GACTCTAGACTGCAGGAGTGAATTAGCCCTTCCA-3′). The PCR product was digested with XbaI and cloned into the XbaI site located downstream of the luciferase gene in pGL3 control. The forward orientation of the insert was checked by sequence analysis.
To construct the m1–m4 mutants, base changes were introduced into pGL3-Nef by mutagenesis PCR (31 (link)). Mutagenic (m) primers EWmut1 (5′-ACAGCAGCTACCAAT
The pRetro-SUPER-shNef vector, which expresses siRNA-Nef under control of the H1 RNA polymerase III promoter was digested with EcoRI and XhoI, and the 314 bp expression cassette was ligated into the EcoRI/XhoI sites of pBluescriptII (KS+) (Stratagene) to produce pBS-siRNA-Nef. Plasmid pRL-CMV (Promega) expresses renilla luciferase under control of the CMV promoter.
