Full length HIV-1 LTR-CAT and the successive deletion constructs at EcoRV, BstNI, HaeIII and MnlI, previously described [
27]; the CMV-CAT and CMV-tat constructs; and the ICP0 expression plasmid were generous gifts from Dr. Mario Estable of Ryerson University, Toronto, Canada, Dr. Lung-ji Chang of the University of Florida and Dr. Paul Kinchington, from the University of Pittsburgh, respectively. The BstΔTAR construct was made by digesting the BstNI construct with KpnI and BglII, blunting the ends with Klenow and then ligating the product with T4 DNA ligase. The SV40-luciferase construct was made by PCR-amplifying the SV40 early promoter out from the pSV-beta galactosidase control vector (Promega, WI) using the primers Fwd- ATTAGATCTGCGCAGCACCATG and Rev- CCAGCAGAGATCCCAAGCTTTTT, digesting the PCR product with the enzymes BglII and HindIII followed by ligation to pGL4.10 (Promega, WI) cut with the same enzymes. The CMV-TAR construct was made by amplifying the TAR region out of HIV-1-LTR with the primers F:TTAGGATCCTTTAGTGAACCGGGTCT and R:GCGGATATCTATTGAGGCTTAAGC, digesting the PCR product with the enzymes BamHI and EcoRV and ligating into the CMV-CAT vector cut with the same enzymes. The 3SpI-TATA construct was created by PCR amplifying the SpI-TATA region of the LTR using the primers 5’ATACTCGAGAGGCGTGGCCT and 5’TACAAGCTTCCAGAGAGACCCAGTA, digesting the PCR product with the restriction enzymes XhoI and HindIII, and ligating it to the pGL4.10 plasmid digested with the same enzymes. SpI deletion mutants were created by overlapping PCR, using the specific DNA oligonucleotides and the corresponding primers listed in Table
1. For the point mutations, the mutations introduced to inactivate the SpI sites (italics) are marked in bold, where G nucleotides were substituted by T:
TT CGTGGC CT
G TT CGGGAC T
GG TT AGTGGC. The oligonucleotide pairs and the corresponding primers used to create these constructs are listed in Table
2. For the deletion constructs, each pair of oligonucleotides was added to a PCR mix containing dNTPs, MgCl2 and DNA polymerase; the reaction was cycled without primers for 5 cycles, with these conditions: (94C, 2’30”; [94C-1’ , 55C-1’ , 72C- 1’45”]X5; 72C-10’), the corresponding primers were then added at a final concentration of 10uM and the tube was cycled for 25 cycles at the same cycling conditions. The PCR product was purified, digested with HindIII and SacI and ligated, using T4 DNA ligase, to the BstΔTAR construct cut with the same enzymes. For the point mutations, the primers used were: Fwd: TGTGAAGCTTTCGGAGGACAGTACTC and Rev TGTGGAGCTCGGATCTGGTCTAAC. To make the point mutation constructs, each of the oligo pairs for constructs K- P (see Table
2) was added at a final concentration of 10 mM to a PCR mix with only the reverse primer, at a final concentration of 1uM and the reaction was amplified for 10 cycles at the following conditions: (94C, 2’30”; [94C-1’ , 55C-1’ , 72C- 1’45”]X10; 72C-10’). Following this, the forward primer was added at a final concentration of 1uM, and the reaction mixture was cycled 25 more times at the same conditions. The PCR product was purified, digested with HindIII and SacI and ligated, using T4 DNA ligase, to the BstΔTAR construct cut with the same enzymes. The sequences of all constructs were confirmed by sequencing with the primer 5’CGCTGGGCCCTTCTTAA, present on the luciferase gene or with the primer 5':CAGCTGAACGGTCTGGTTATAG present on the CAT gene. CMV-Renilla luciferase (Promega, Madison, WI) was used as transfection control.
Table 1
SpI deletion construct oligos and primers
1 | Del 1, del 2 | 5’TCGGAGGACAGTACTCCGACCCGGTCGAAGGGAGGCGTGGCCTGAGCCCTCAGATCCTGCATATAA GC |
And |
5’GAGCCCTCAGATCCTGCATATAA GCAGCTGCTTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCCGAGC |
2 | Del 1 | 5’TCGGAGGACAGTACTCCGACCCGGTCGAAGGGAGGCGTGGCCTGGGCGGGACTGAGCCCTCAGATCCTGCATATAA GC |
And |
5’GAGCCCTCAGATCCTGCATATAA GCAGCTGCTTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCCGAGC |
3 | Del 2 | 5’TCGGAGGACAGTACTCCGACCCGGTCGAAGGGAGGCGTGGCCTTGGGGAGTGGCGAGCCCTCAGATCCTGCATATAA |
And |
| | 5’GAGCCCTCAGATCCTGCATATAA GCAGCTGCTTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCCGAGC |
Table 2
SpI inactivating point mutation construct oligos and primers
mut | Wt | wt | K |
mut | Wt | mut | L |
mut | Mut | wt | M |
wt | Mut | wt | N |
wt | Mut | mut | O |
wt | Wt | mut | P |
Construct | Oligos used (Oligo 2 is the same for all constructs) |
K | Oligo 1: TCGGAGGACAGTACTCCGACCCGGTCGAAGGGATTCGTGGCCTGGGCGGGACTGGGGAGTGGCGAGCCCTCAGATCCTGCATATAA |
Oligo 2: GAGCCCTCAGATCCTGCATATAA GCAGCTGCTTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATC CGAGC |
L | Oligo 1: TCGGAGGACAGTACTCCGACCCGGTCGAAGGGATTCGTGGCCTGGGCGGGACTGGTTAGTGGCGAGCCCTCAGATCCTGCATATAA |
M | Oligo 1: TCGGAGGACAGTACTCCGACCCGGTCGAAGGGATTCGTGGCCTGTTCGGGACTGGGGAGTGGCGAGCCCTCAGATCCTGCATATAA |
N | Oligo 1: TCGGAGGACAGTACTCCGACCCGGTCGAAGGGAGGCGTGGCCTGTTCGGGACTGGGGAGTGGCGAGCCCTCAGATCCTGCATATAA |
O | Oligo 1: TCGGAGGACAGTACTCCGACCCGGTCGAAGGGAGGCGTGGCCTGTTCGGGACTGGTTAGTGGCGAGCCCTCAGATCCTGCATATAA |
P | Oligo 1: TCGGAGGACAGTACTCCGACCCGGTCGAAGGGAGGCGTGGCCTGGGCGGGACTGGTTAGTGGCGAGCCCTCAGATCCTGCATATAA |