Download The full-length HIV-1 molecular clone pLAI 61 was used to produce

Supplementary information
Construction of lhRNA-expression constructs
The full-length HIV-1 molecular clone pLAI
1
was used to produce wild-type virus and to
study inhibition by lhRNAs directed either against the tat, rev or nef sequences. Nucleotide
numbers refer to the position on the genomic HIV-1 RNA transcript, with +1 being the
capped G residue. Supplementary Table 1 lists all oligonucleotides used in this study. The tat
exon 1 was amplified by PCR on pLAI with primers NotI-WdV005 and WdV002; tat exon 2
with primers WdV007 and NotI-WdV004; rev exon 1 with primers NotI-WdV001 and
WdV002; rev exon 2 with primers WdV003 and NotI-WdV004. The tat exon 1 and 2 PCR
products were mixed in equimolar amounts and subjected to a second PCR with NotIWdV005 and NotI-WdV008. Similarly, rev exon 1 and 2 PCR products were reamplified with
primers NotI-WdV001 and NotI-WdV004. The 276 bp tat cDNA fragment and 366 bp rev
cDNA fragment were ligated into the pGEM-T vector (Promega), yielding pGEM-T-tat and
pGEM-T-rev, respectively. The 653 bp HIV-1 nef fragment (position 8390 – 9010) was
amplified by PCR on pLAI DNA using primers NotI-WdV011 and NotI-WdV012 and cloned
into pGEM-T, creating pGEM-T-nef.
Construction of pEF1α-tat. The human elongation factor 1 α (EF1α) promoter, the
Gateway (Invitrogen) cassette and the bovine growth hormone gene polyadenylation signal
(BGH-polyA) of plasmid pEF5/FRT/V5-DEST (Invitrogen) were removed by HindIII and
SphI digestion. A polylinker DNA fragment containing HindIII, NotI, SacI, AvrII, XhoI,
SgrAI, PacI and SphI restriction sites was created by self-annealing of oligos WdV022 and
WdV023, WdV024 and WdV025. The resulting DNA fragment was cloned in the HindIII and
SphI sites of pEF5/FRT/V5-DEST, yielding pWdV06.3. The EF1α promoter was PCR
amplified from pEF5/FRT/V5-DEST with primers HindIII-WdV047 and WdV061. A 300 bp
antisense tat (as-tat) DNA fragment was PCR amplified from pGEM-T-tat with primers
WdV049 and NotI-WdV050. The EF1α and as-tat fragments were mixed and subjected to a
second PCR with primers HindIII-WdV047 and NotI-WdV050, and ligated into the HindIII
and NotI sites of pWdV06.3 to yield pWdV17. The BGH-polyA was amplified by PCR on
pEF5/FRT/V5-DEST with primers WdV053 and PacI-WdV054. A 300 bp sense tat (s-tat)
DNA fragment was PCR amplified from pGEM-T-tat with primers AvrII-WdV051 and
WdV52. The s-tat and BGH-polyA fragments were mixed and re-amplified by PCR with
AvrII-WdV051 and PacI-WdV054 and cloned in the AvrII and PacI sites of pWdV17,
yielding pEF1α-tat (Fig. 3).
Construction of pEF1α-rev. The EF1α promoter was amplified by PCR on
pEF5/FRT/V5-DEST with primers HindIII-WdV047 and WdV055. A 300 bp anti-sense rev
(as-rev) fragment was PCR amplified from pGEM-T-rev with primers WdV056 and NotIWdV057. Both EF1α and as-rev fragments were mixed and subjected to a second PCR with
HindIII-WdV047 and NotI-WdV057. The resulting fragment was ligated into the HindIII and
NotI sites of pWdV06.3, yielding pWdV19. The BGH-polyA was amplified by PCR on
pEF5/FRT/V5-DEST with primers WdV60 and PacI-WdV054. A 300 bp sense rev (s-rev)
was PCR amplified from pGEM-T-rev with primers AvrII-WdV058 and WdV059. The s-rev
and BGH-polyA fragments were mixed and re-amplified by PCR with AvrII-WdV058 and
PacI-WdV054 and the resulting fragment was ligated in the AvrII and PacI sites of pWdV19
to yield pEF1α-rev (Fig. 3).
Construction of pEF1α-nef1. The EF1α promoter was PCR amplified from
pEF5/FRT/V5-DEST with primers HindIII-WdV047 and WdV061. A 300 bp anti-sense nef1
(as-nef1) fragment was amplified by PCR on pGEM-T-nef with primers WdV062 and NotIWdV063. The EF1α and as-nef1 fragments were fused by PCR with primers HindIIIWdV047 and NotI-WdV063 and cloned into the HindIII/NotI sites of pWdV06.3 to yield
pWdV21. The BGH-polyA was amplified by PCR on pEF5/FRT/V5-DEST with primers
WdV066 and PacI-WdV054. A 300 bp sense nef1 (s-nef1) fragment was PCR amplified from
pGEM-T-nef with primers AvrII-WdV064 and WdV065. Both BGH-polyA and s-nef
fragments were mixed and re-amplified by PCR with primers AvrII-WdV064 and PacIWdV054 and cloned into likewise digested pWdV21, yielding pEF1α-nef1 (Fig. 3).
Construction of pEF1α-GFP. A DNA fragment comprising the hrGFP gene was PCR
amplified from PSXrabpA (Stratagene) with primers attB1-WdV020 and attB2-WdV021. The
hrGFP was recombined into pEF5/FRT/V5-DEST using the Gateway technology, resulting in
the hrGFP gene expression vector pHY01. The EF1α promoter was amplified by PCR on
pEF5/FRT/V5-DEST with primers SphI-WdV034 and WdV035. A second PCR was
performed to amplify the hrGFP fragment from pHY01 with primers WdV036 and BamHIWdV037. Both EF1α and hrGFP fragments were mixed and subjected to a PCR with SphIWdV034 and BamHI-WdV037 and the resulting PCR product was ligated into SphI and
BamHI sites of pUC19 to yield pWdV09. An antisense fragment from hrGFP (as-hrGFP)
gene was amplified by PCR on pHY01 with primers BamHI-WdV038 and WdV039. A
second PCR was performed to amplify an SV40-polyA (SV40-pA) fragment from plasmid
pPUR (Clontech) using primers WdV040 and EcoRI-WdV041. as-hrGFP and SV40-pA
fragments were mixed and subjected to PCR with BamHI-WdV038 and EcoRI-WdV041. The
resulting fragment was ligated into the BamHI and EcoRI sites of pWdV09, yielding
pWdV10. pWdV10 was digested with SphI and the fragment containing the GFP inverted
repeat was ligated in the likewise digested pHY01 plasmid to yield pEF1α-GFP (Fig. 3).
Construction of pLTR-tat, pLTR-nef1, p7tetO-tat and p7tetO-nef1. The HIV-1 LTR
was PCR amplified from pBlue3’LTR-luc
2
with primers SalI-PKF5 and HindIII-PKR5;
7tetO was amplified from the CMV-7tetO promoter/luciferase reporter construct pUHC13-3 3
with primers SalI-PKF6 and HindIII-PKR6. Both LTR and 7tetO amplicons were cloned in
the SalI and HindIII sites of pBluescript SK- (Stratagene) to yield pBlue-LTR and pBlue7tetO, respectively. To create the lhRNA tat and nef1, approximately 300 nt as-tat and as-nef1
fragments were PCR amplified from pEF1α-tat and pEF1α-nef1 with primers HindIII-PKF4
and NotI-PKR2, and cloned into the HindIII and NotI sites of pBlue-LTR and pBlue-7tetO,
resulting in pLTRas-tat, pLTRas-nef1, p7tetOas-tat and p7tetOas-nef1. Two oligos PKF8 and
PKR13 were self-annealed to create a polylinker NotI-MunI-BsaBI-XbaI-AvrII-SacIIBamHI-EcoRV-BbrPI-SacII that was ligated in the NotI and SacII sites of the above pLTRand 7tetO-containing plasmids. The intron of the EF1α promoter was amplified from
pWdV65 with primers MunI-PKF13 and XbaI-PKR9 and ligated in the MunI and XbaI sites
of the polylinker. The s-tat and s-nef1 fragments, together with the BGH-polyA, were
amplified from pEF1α-tat and pEF1α-nef1 with primers PKF2 and BamHI-PKR4 and cloned
into the AvrII and BamHI sites downstream of the EF1α intron, resulting in pLTR-tat, pLTRnef1, p7tetO-tat, and p7tetO-nef1 (Fig. 3). Vector pLTR-nef1 (Fig. 3) was created by
insertion of the 554 nt HindIII fragment of pLAI (, 76 - 630) into the corresponding site of
pLTR-nef1. The Ψ fragment includes the HIV-1 polyA signal, primer-binding site,
dimerisation signal, splice donor site and packaging signal. Plasmid pLTR has been
described previously 4.
Construction of pT7-nef2 and pT7sh-nef. Plasmid pGEM-T-nef was PCR amplified
with primers SacI-T7p-PKF16 and asT7p-XbaI-PKR16. The resulting nef2 PCR product that
contains convergent T7 promoters and terminators was cloned in pCR2.1-TOPO (Invitrogen)
to yield pT7-nef2 (Fig. 3). Plasmid pT7sh-nef was constructed by self-annealing of two
complementary oligos NcoI-PKF21 and BamHI-PKR21 that were cloned into the NcoI
andBamHI sites of pT7-luc (5,6, a kind gift of Dr. P. Midoux, Centre de Biophysique
Moleculaire, Orleans, France), replacing the Photinus pyralis luciferase (firefly luciferse, FL)
cDNA. Plasmid pcDNA3-T7pol, expressing bacteriophage T7 polymerase (pT7-pol, a kind
gift of Dr. Jean-Marc Jacque, University of Massachusetts Medical School, USA), and
pH1sh-nef have been described previously 7,8.
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