Table 1. Oligonucleotide primers used for the HIV-1 50-UTR mutagenesis DNA wt wt A1 r A1 f A2 f A3 r A3 f A4 r A4 f A5 r A5 f A6 r A6 f A7 r A7 f A8 r A8 f U1 r U1 f U2 f U3 r U3 f U4 r U4 f U5 r U5 f U6 r U6 f U7 r U7 f U8 r U8 f A2-LF f U2-LF f Primer TA033 TA056 TA057 TA058 TA059 TA060 TA061 TA062 TA063 TA064 TA065 TA066 TA067 TA068 TA069 TA070 TA071 TA122 TA123 TA104 TA232 TA233 TA234 TA235 TA082 TA083 TA105 TA067 TA084 TA085 TA086 TA087 TA110 TA231 Nucleic Acids Research, 2011, Vol. 39, No. 12 5235 Primer sequence 50!30 CCCCTCGAGTAATACGACTCACTATAGGGTCTCTCTGGTTAGACC GGGTCATGAGTCGACCCCCTGGCCTTAACCG GGCCATGGTTCCCTAGTTAGCCAG AGGGAACCATGGCCACTGCTTAAGCCTCA AATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGACCATGGCGTCTGTTGTGTGACTCTGG AAAAGCCATGGTCTGAGGGATCTCTAGTTACC CAGACCATGGCTTTTAGTCAGTGTGGAAAATC CCCATGGTTTCGCTTTCAAGTCCC GAAAGCGAAACCATGGGAAACCAGAGGAGC GCCCATGGTCGAGAGAGCTCCTCTGGTTTCCC CTCTCGACCATGGGCAGGACTCGGCTTGCTGAAGC ACTCACCAGTCGCCTCCATGGTCCTCGCCTCTTGCCGTGCGC GGAGGCCGACTGGTGAGTACG CCATGGTGGCGTACTCACCAGTCGCCTCCCC GAGTACGCCACCATGGAAAATTTTGACTAGCGGAGGC CTCTCTCCATGGTCCTTCTAGCCTCCGCTAG AGAAGGACCATGGAGAGAGATGGGTGCGAGAGCG GGCCTAGGTTCCCTAGTTAGCCAG AGGGAACCTAGGCCACTGCTTAAGCCTCA AATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGACCTAGGCGTCTGTTGTGTGACTCTGG AAAAGCCTAGGTCTGAGGGATCTCTAGTTACC CAGACCTAGGCTTTTAGTCAGTGTGGAAAATC CCCTAGGTTTCGCTTTCAAGTCCC GAAAGCGAAACCTAGGGAAACCAGAGGAGC GCCCTAGGTCGAGAGAGCTCCTCTGGTTTCCC CTCTCGACCTAGGGCAGGACTCGGCTTGCTGAAGC ACTCACCAGTCGCCTCCTAGGTCCTCGCCTCTTGCCGTGCGC GGAGGCCGACTGGTGAGTACG CCTAGGTGGCGTACTCACCAGTCGCCTCCCC GAGTACGCCACCTAGGAAAATTTTGACTAGCGGAGGC CTCTCTCCTAGGTCCTTCTAGCCTCCGCTAG AGAAGGACCTAGGAGAGAGATGGGTGCGAGAGCG AAGTAGTGTGTGACCATGGCGTCTGTTGTGTGGACTCTGGTAACTAGAGATCCC CTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGACCTAGGCGTCTGTTGTGTGGACT CTGGTAACTAGAGATCCC f = forward, r = reverse primer. Nucleotide insertions are indicated in bold italics. seeded into 24-wells plates and transfected the next day with calcium phosphate-precipitated luciferase con- structs in the presence of pcDNA3-Tat or pcDNA3 (67,69). pRL-CMV expressing Renilla luciferase was co-transfected as internal control. pBlueScript was used to make a total amount of 1 mg DNA for each transfec- tion. Cells were washed with PBS 2 days after transfection and lysed in 125 ml of passive lysis buffer provided by the Dual-luciferase Reporter Assay System (Promega). The ﬁreﬂy and Renilla luciferase activity was determined according to the manufacturer’s protocol. The ﬁreﬂy luciferase activity was normalized to the Renilla luciferase activity. RNA quantiﬁcation C33A cells were transfected at 50% conﬂuence in a 6-well plate with 3 mg CaPO4-precipitated luciferase reporter construct as described. Two days post-transfection RNA was isolated with Trizol as described by the manufacturer. The RNA pellet was dissolved in 50 ml H2O and DNase treated with 1 ml Turbo DNase (Ambion) for 30 min at 37 C. The reaction was stopped by phenol/chloroform ex- traction with subsequent ethanol precipitation. The RNA was dissolved in 25 ml H2O and analysed and quantiﬁed on an ethidium bromide-stained agarose gel (1.5%). Primer extension reactions were performed on equal amounts of total RNA. The oligonucleotide primer R368 (50-TCCCCCGCTTAATACTGACGCT) was end-labelled with [g-32P]-dATP using T4 polynucleotide kinase for 2 h at 37 C and puriﬁed using NucAway spin columns (Ambion). Subsequently, the primer was annealed to the RNA and reversed transcribed with Thermoscript reverse transcriptase (Thermoscript kit, Invitrogen). The reaction was stopped with 7 ml of 1 M NaOH for 15 min at 70 C and subsequent neutralization with 7 ml of 1 M HCl. The cDNA products were ethanol-precipitated, dissolved in formamide-containing loading buffer and applied to a denaturing polyacrylamide gel. Products were analysed using a PhosphorImager (Molecular Dynamics). Sodium dodecyl sulphate–polyacrylamide gel electophoresis and western blot analysis After determining the ﬁreﬂy and Renilla activities in lysates from transfected cells, equal amounts of Renilla luciferase activity were applied to a 12% sodium dodecyl